Men protein, gst2, rab-rp1, csp, f-box protein lilina/fbl7, abc50, coronin, sec61 alpha, or vhappa1-1, or homologous proteins involved in the regulation of energy homeostasis

ABSTRACT

The present invention discloses Men protein, GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1 and homologous proteins regulating the energy homeostasis and the metabolism of triglycerides, and polynucleotides, which identify and encode the proteins disclosed in this invention. The invention also relates to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases and disorders, for example, but not limited to, metabolic diseases such as obesity as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea.

This invention relates to the use of nucleic acid sequences encodingmalic enzyme (referred to as Men protein), Glutathione S-transferase 2(referred to as GST2), Rab-related protein 1 (referred to as Rab-RP1),Cysteine string protein (referred to as Csp), CG11033 (referred to asF-box protein Lilina/FBL7), CG1703 (ABCF1, TSAP; referred to as ABC50),coro (referred to as coronin), Sec61 alpha, and VhaPPA1-1, or tomammalian, particularly human Men protein, GST2, Rab-RP1, Csp, F-boxprotein Lilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1homologous proteins (for example, NADP-dependent cytosolic malic enzyme1 (ME1), NADP-dependent mitochondrial malic enzyme 3 (ME3),NAD(+)-dependent mitochondrial malic enzyme 2 (ME2), hematopoieticprostaglandin D2 synthase (PGDS), RAB32, RAB38, RAB7, cysteine stringprotein 2, gamma cysteine string protein, Beta cysteine string protein,F-box and leucine-rich repeat protein 11 (FBL11), JEMMA protein, PHDfinger protein 2, protein with GenBank Accession Number AAC83407, ABC50(TNF-alpha stimulated ABC protein), coronin 1B, coronin 1C,clipinE/coronin 6 type B, coronin 2A, coronin 2B, Sec61 alpha form 2,Sec61 alpha form 1, and vacuolar ATP synthase 21 kDa proteolipidsubunit), and the polypeptides encoded thereby and effectors thereof andto the use thereof in the diagnosis, study, prevention, and treatment ofdiseases and disorders related to body-weight regulation, for example,but not limited to, metabolic diseases such as obesity as well asrelated disorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, and sleep apnea.

There are several metabolic diseases of human and animal metabolism,eg., obesity and severe weight loss, that relate to energy imbalancewhere caloric intake versus energy expenditure is imbalanced. Obesity isone of the most prevalent metabolic disorders in the world. It is stilla poorly understood human disease that becomes as a major health problemmore and more relevant for western society. Obesity is defined as a bodyweight more than 20% in excess of the ideal body weight, frequentlyresulting in a significant impairment of health. It is associated withan increased risk for cardiovascular disease, hypertension, diabetes,hyperlipidaemia and an increased mortality rate. Besides severe risks ofillness, individuals suffering from obesity are often isolated socially.

Obesity is influenced by genetic, metabolic, biochemical, psychological,and behavioral factors. As such, it is a complex disorder that must beaddressed on several fronts to achieve lasting positive clinicaloutcome. Obese individuals are prone to ailments including: diabetesmellitus, hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancers of the reproductiveorgans, and sleep apnea.

Since obesity is not to be considered as a single disorder but aheterogeneous group of conditions with (potential) multiple causes, itis also characterized by elevated fasting plasma insulin and anexaggerated insulin response to oral glucose intake (Koltermann, J.Clin. Invest 65, 1980, 1272-1284) and a clear involvement of obesity intype 2 diabetes mellitus can be confirmed (Kopelman, Nature 404, 2000,635-643).

The molecular factors regulating food intake and body weight balance areincompletely understood. Even if several candidate genes have beendescribed which are supposed to influence the homeostatic system(s) thatregulate body mass/weight, like leptin, VCPI, VCPL, or the peroxisomeproliferator-activated receptor (PPAR)-gamma co-activator, the distinctmolecular mechanisms and/or molecules influencing obesity or bodyweight/body mass regulations are not known. In addition, severalsingle-gene mutations resulting in obesity have been described in mice,implicating genetic factors in the etiology of obesity. (Friedman andLeibel, 1990, Cell 69: 217-220). In the obese mouse model, a single genemutation (obese) results in profound obesity, which is accompanied bydiabetes (Friedman et. al., 1991, Genomics 11: 1054-1062).

Therefore, the technical problem underlying the present invention was toprovide for means and methods for modulating (pathological) metabolicconditions influencing body-weight regulation and/or energy homeostaticcircuits. The solution to said technical problem is achieved byproviding the embodiments characterized in the claims.

Accordingly, the present invention relates to genes with novel functionsin body-weight regulation, energy homeostasis, metabolism, and obesity.The present invention discloses specific genes involved in theregulation of body-weight, energy homeostasis, metabolism, and obesity,and thus in disorders related thereto such as eating disorder, cachexia,diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancer,e.g. cancers of the reproductive organs, and sleep apnea. The presentinvention describes the human Men protein, GST2, Rab-RP1, Csp, F-boxprotein Lilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1 (hereinrefered to as 'proteins of the invention) homologous genes and proteinsencoded thereby as being involved in those conditions mentioned above.

The term “GenBank Accession number” relates to National Center forBiotechnology Information (NCBI) GenBank database entries (Benson et al,Nucleic Acids Res. 28, 2000, 15-18).

Men

The Drosophila Men gene with GadFly Accession Number CG10120 encodes fora malate dehydrogenase (oxaloacetate decarboxylating) (NADP+)(EC:1.1.1.40). Men is highly conserved and might be differentiallyspliced in addition to other malic enzymes like Mdh (GenBank AccessionNumber AE003759). We found that Drosophila Men protein is mosthomologous to human and mouse Men proteins. Men is the structural genefor malic enzyme, which is identical to (S)-Malate: NADP+ oxidoreductase(MEN). The enzyme is known to provide NADPH for lipogenesis; NADPHlevels in fly larvae are increased by dietary carbohydrate and decreasedby dietary lipid. Highest specific activity found in larval fat bodyand, among cellular fractions, in the cytosol (Geer et al. B. W. (1979)Biochem Genet 17(9-10):867-879).

The human Men gene encodes for the cytosolic form of an enzyme of thecitrate cycle (Malate+NAD⁺→Oxalacetate+NADH+H⁺) that is localised inmitochondria and is NAD+ coupled. In addition, there is also amitochbndrial form of malate dehydrogenase that is NADH coupled.Alternatively, it might encode for the NADP⁺ dependent malic enzyme thatcatalyzes Malate+NADP⁺→Pyruvate+CO₂+NADPH.

GST2

GSH-dependent prostaglandin D(2) synthase (GST2) enzymes represent theonly vertebrate members of class Sigma glutathione S-transferases (GSTs)identified to date (see, Kanaoka et al., 2000, Eur. J. Biochem.267:3315-3322). Orthologous human and rat GSH-dependent GST2 were bothshown to catalyse specifically the isomerization of prostaglandin (PG)H(2), a common precursor of various prostanoids, to produce PGD2 as amajor prostanoid in a variety of tissues (review, see, for example,Urade & Hayaishi Vitam Horm 2000;58:89-120). Each transferase alsoexhibited GSH-conjugating and GSH-peroxidase activities (Jowsey et al.,Biochem J 2001 359(Pt 3):507-16).

PGD2 has various functions in the peripheral tissues, such as preventionof platelet aggregation and induction of vasodilatation andbronchoconstriction. PGD2 is released from mast cells stimulated byvarious immunological stimulants and functions as a lipid mediator inallergy and inflammation. PGD2 is further converted to 9 alpha, 11beta-PGF2 or the J series of prostanoids. The J series of PGs were foundto have an antiproliferative effect against tumor cells (see, forexample, Fukushima et al, 1994, Ann. N.Y. Acad. Sci 744:161-165]. A PGJ2metabolite, 15-deoxy-D12,14-PGJ2, which promotes adipocytedifferentiation, was identified as a natural ligand for the peroxisomeproliferator-activated receptor (PPAR) gamma (see, for example, Klieweret al. 1995, Cell 83:813-819). The ligand activation of PPAR gamma wasfound to regulate macrophage and monocyte functions (see, for example,Huang et al., 1999, Nature 400:378-382).

Two types of PGD synthase exist, the lipocalin-type enzyme and thehematopoietic enzyme. Hematopoietic PGD synthase is widely distributedin the peripheral tissues and localized in the antigen-presenting cells,mast cells, and megakaryocytes. The hematopoietic enzyme is the firstrecognized vertebrate homolog of the sigma class of glutathioneS-transferase (see, Kanaoka et al., Eur J Biochem 2000 267(11):3315-22).X-ray crystallographic analyses and generation of gene-knockout andtransgenic mice for each enzyme have been performed.

Hepatic glutathione S-transferase activity was studied in obese mice(Wolff & Suber, Proc Soc Exp Biol Med 1986 181(4):535-41). It was foundthat the hepatic glutathione S-transferase activity of yellow Avy/a (YSX VY) F-1 hybrid female mice was decreased compared to the activitymeasured black a/a female mice which was associated with the obesity ofthe yellow mice.

RabRP1

Rab proteins constitute a family of GTP-binding proteins that arelocated in distinct intracellular compartments and play a role in theregulation of vesicular trafficking, including exocytosis andendocytosis (see, for review, Armstrong, Int J Biochem Cell Biol 200032(3):303-7 J). More than 50 mammalian Rab proteins are known, many withtransport step-specific localisation. Through their effectors, RabGTPases regulate vesicle formation, actin- and tubulin-dependent vesiclemovement, and membrane fusion. A number of Rab GTPases are conservedfrom yeast to humans. Yeast mutations in Rab gene homologs cause defectsin vesicular transport similar to those observed in beige (bg) mice, themurine Hermansky-Pudlak syndrome model.

Rab-related small GTP-binding protein (Rab38) has been localized to thelung, especially alveolar type II cells and bronchial epithelial cells,suggesting a role in vesicular transport in terminal airway epithelium(see Osanai et al. Am J Pathol 2001 158(5):1665-75). In addition, Rab38is showing a predominant mRNA expression in melanocytes, a cell-specificexpression pattern likely related to melanosomal transport and docking(see Jager et al. Cancer Res 2000;60(13):3584-91). Among the family ofrab proteins, rab38 has a unique COOH terminus which would allowposttranslational farnesylation and palmitoylation, lipid modificationsnormally occurring in ras proteins but not in other rab proteins (seeJager et al. 2000, supra).

A review by Cormont and Le Marchand-Brustel discusses the role of smallG-proteins in the regulation of glucose transport (see, Mol Membr Biol2001 July-September;18(3):213-20A). They discuss that insulin increasesthe rate of glucose transport into fat and muscle cells by stimulatingthe translocation of intracellular Glut 4-containing vesicles to theplasma membrane, along with an increase in the amount of thefacilitative glucose transporter Glut 4 at the cell surface, allowingfor an enhanced glucose uptake. This process requires a continuouscycling through the early endosomes, a Glut 4 specific storagecompartment and the plasma membrane. The main effect of insulin is toincrease the rate of Glut 4 trafficking from its specific storagecompartment to the plasma membrane. The whole phenomenon involves signaltransduction from the insulin receptor, vesicle trafficking (sorting andfusion processes) and actin cytoskeleton modifications, which are allsupposed to require small GTPases.

A member of the Rab 3 subfamily of small GTP-binding proteins, Rab 3D,in rat adipose cells, has been postulated to be involved ininsulin-stimulated GLUT4 exocytosis (Guerre-Millo et al. Biochem J.1997;321 (Pt 1):89-93). Rab 3D is overexpressed in adipose cells ofobese (fa/fa) Zucker rats, in a tissue- and isoform-specific manner. Thepathophysiological significance of this defect remains elusive whichcould form the molecular basis for altered adipose secretory function inobesity.

CSP

Cysteine-string protein (Csp) is a major synaptic vesicle and secretorygranule protein first discovered in Drosophila and Torpedo (for review,see, for example, Chamberlain & Burgoyne, 2000, J Neurochem 200074(5):1781-9 RD), and were subsequently identified from Xenopus,Caenorhabditis elegans, and mammalian species. Studies from the nullmutant in Drosophila have shown that Csp is required for viability ofthe organism. It has been also shown that Csp plays a key role inneurotransmitter release. Amorphic Drosophila mutations have beenisolated which affect the larval neuromuscular junction and areconditional temperature sensitive paralytic, conditional temperaturesensitive neurophysiology defective and recessive semi-lethal.Furthermore, other studies have directly implicated Csp in regulatedexocytosis in mammalian neuroendocrine and endocrine cell types, and itsdistribution suggests a general role in regulated exocytosis. Cspspossess a cysteine-string domain that is highly palmitoylated andconfers membrane targeting. In addition, Csps have a conserved “J”domain that mediates binding to an activation of the Hsp70/Hsc70chaperone ATPases. Targets for Csp include the vesicle proteinVAMP/synaptobrevin and the plasma membrane protein syntaxin 1.

It has been shown that the cysteine-string protein is associated withthe plasma membrane in 3T3-L1 adipocytes but not with intracellularGlut4-storage vesicles. Csp1 interacts with the t-SNARE protein syntaxin4 which is an important mediator of insulin-stimulated fusion with theplasma membrane, suggesting that Csp1 may play a regulatory role in thisprocess. In contrast, syntaxin 1A binds to both Csp isoforms (Csp1 andCsp2), with higher affinity for the Csp2 protein (see, Chamberlain etal., 2001, J Cell Sci;114(Pt 2):445-55).

F-Box

The Drosophila gene CG11033 encodes for a F-box-like protein involved inneuropeptide signaling that is required for normal circadian locomotorrhythms in Drosophila. Interpro analysis of this gene reveals cytochromec family heme-binding site, an F-box protein Lilina/FBL7 domain, CXXCzinc finger and a glycin-rich region domains. We found that F-boxprotein Lilina/FBL7 is most homologous to human F-box proteinLilina/FBL7 protein (GenBank Accession Number NP_(—)036440.1) which wasrecently cloned by Ilyin et al., 2000 (Genomics 67(1):40-47). F-boxproteins are components of the SCF ubiquitin-protein ligase complexwhich functions in several biological processes like cell cycle control,apoptosis, transcription, and signal transduction. It has been shownthat the SCF ubiquitin-protein ligase complex is essential for theNF-kappaB, Wnt/Wingless, and Hedgehog signaling pathways (Maniatis T.,(1999) Genes Dev. 13(5):505-510), signaling pathways that are involvedin metabolism.

ABC50

The ABC50 is a member of the ATP-binding cassette (ABC) proteins. Unlikethe majority of ABC proteins, which are membrane-associatedtransporters, ABC50 associates with the ribosome in an ATP-dependentmanner (see, Tyzack et al., J. Biol. Chem. 275: 137-45). ABC 50 has beenshown to interact with eukaryotic initiation factor 2 (elF2), whichplays a key role in the process of translation initiation and in itscontrol. ABC50 is related to GCN20 and eEF3, two yeast ABC proteins thatare not membrane-associated transporters and are instead implicated inmRNA translation and/or its control. Therefore, ABC50 is considered asan ABC protein with a likely function in mRNA translation, whichassociates with elF2 and with ribosomes. A role of ABC50 in theenhancement of protein synthesis has been postulated that followsTNF-alpha treatment of synoviocytes and thus participates in theinflammatory processes mediated by this cytokine (Richard et al.Genomics, 1998, 53:137-45).

Coronin

Coronin belongs to a family of actin-associated proteins and was firstisolated from Dictyostelium, but similar proteins have been identifiedin many species and individual cell types (for review, see de Hostos,Trends Cell Biol 1999 9(9):345-350). Coronin is an actin-bindingprotein, which contains WD (Trp-Asp) repeats and a coiled-coil motif,and plays a role in regulating organization of the actin cytoskeletalnetwork. Coronin localizes to the cell periphery, is involved inlamellipodium extension, and has an implicated role in cytokinesis, cellmotility and phagocytosis. During phagocytosis coronin is recruitedtogether with PI 3-kinase to membranes of nascent and early phagosomesco-localizing with the actin cytoskeleton, confirming that coronincontributes to phagocytosis (see, for example, Didichenko et al., FEBSLett. 2000 24;485(2-3):147-152). Although the existence of coronin inhigher eukaryotes has been reported, its function in vertebrate cellshas not been elucidated.

Sec61 Alpha

The Sec61 complex is a central component of the endoplasmic reticulum(ER) translocation site (translocon). The complex consists of threesubunits: Sec61 alpha, Sec61 beta and Sec61 gamma, at least two of which(alpha and beta) are adjacent to nascent proteins during membraneinsertion. Sec61 alpha functions as the major component of atransmembrane channel formed by oligomers of the Sec61 complex. Thischannel is the site of secretory protein translocation and membraneprotein integration at the ER membrane. Sec61 alpha is a polytopicintegral membrane protein (see, for example, Knight and High, (1998)Biochem J 331 (Pt 1):161-167). Sec61 alpha has SecY protein domains.Sec61 alpha was reported to interact with Grp170, Grp94, BiP/Grp78,calreticulin, and protein disulfide isomerase (see, Dierks et al.,(1996) EMBO J 15(24):6931-6942).

Mitchell et al. describe that Apoprotein B100 has a prolongedinteraction with the translocon during which its lipidation andtranslocation change from dependence on the microsomal triglyceridetransfer protein to independence (Proc Natl Acad Sci U S A. 199895(25):14733-8). Pariyarath et al. discuss the co-translationalinteractions of apoprotein B with the ribosome and translocon duringlipoprotein assembly or targeting to the proteasome (see, J Biol Chem.2001 Jan. 5;276(1):541-50).

VhaPPA1-1

The Drosophila VhaPPa1-1 encodes for a hydrogen-transporting two-sectorATPase which is a component of the hydrogen-transporting ATPase Vodomain. Intrapro analysis reveals vacuolar ATP synthase 16 kD subunitand ATP synthase subunit C protein domains. VhaPPa1-1 is most homologousto mouse vacuolar proton-translocating ATPase 21 kDa subunit and tohuman ATPase, H⁺ transporting, lysosomal 21 KD subunit. Vacuolar ATPasesare involved in the lysosomal transport and metabolism of lipoproteinslike LDL (see, for example, U.S. Pat. No. 6,107,462). The proteolipiddomain of vacuolar H(+)-ATPase (V-ATPase) plays a major role in H⁺transport in microvesicles and other acidic organelles. Nishigori et alGenomics 1998 Jun. 1;50(2):222-8 have cloned the second humanproteolipid of the V-ATPase (designated hATP6F), a homologue of theSaccharomyces cerevisiae proteolipid VMA16, which is an essentialsubunit of yeast V-ATPase. hATP6F is a hydrophobic protein with fiveputative transmembrane segments, having 61% amino acid identity and 83%similarity to the yeast protein, except in the N-terminus, and containsa conserved glutamic acid residue (Glu98) that is essential forH⁺-transporting activity. The epitope-tagged 23-kDa protoelipid waslocalized in endomembrane organelles in CHO cells, as expected for acomponent of a vacuolar-type proton pump (Sun-Wada et al. Gene 2001274(1-2):93-99).

So far, it has not been described that malic enzyme (referred to as Menprotein), Glutathione S-transferase 2 (referred to as GST2), Rab-relatedprotein 1 (referred to as Rab-RP1), Cysteine string protein (referred toas Csp), CG11033 (referred to as F-box protein Lilina/FBL7), CG1703(ABCF1, TSAP; referred to as ABC50), coro (referred to as coronin),Sec61 alpha, and VhaPPA1-1, or human Men protein, GST2, Rab-RP1, Csp,F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1homologous proteins are involved in the regulation of energy homeostasisand body-weight regulation and related disorders, and thus, no functionsin metabolic diseases and other diseases as listed above have beendiscussed. In this invention we demonstrate that the correct gene doseof Men protein, GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50,coronin, Sec61 alpha, or VhaPPA1-1 is essential for maintenance ofenergy homeostasis. A genetic screen was used to identify that mutationof Men protein, GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50,coronin, Sec61 alpha, or VhaPPA1-1 homologous genes cause obesity,reflected by a significant increase of triglyceride content, the majorenergy storage substance.

Polynucleotides encoding a protein with homologies to Men protein, GST2,Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, orVhaPPA1-1 are suitable to investigate diseases and disorders asdescribed above. Molecules related to Men protein, GST2, Rab-RP1, Csp,F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1 aresuitable for providing new compositions useful in diagnosis, treatment,and prognosis of diseases and disorders as described above.

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure.

In this invention we particularly refer to Malic enzyme (referred to asMen protein), Glutathione S-transferase 2 (referred to as GST2),Rab-related protein 1 (referred to as Rab-RP1), Cysteine string protein(referred to as Csp), CG11033 (referred to as F-box proteinLilina/FBL7), CG1703 (ABCF1, TSAP; referred to as ABC50), coro (referredto as coronin), Sec61 alpha, and VhaPPA1-1, and Men protein, GST2,Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha,and VhaPPA1-1 homologous proteins (for example, NADP-dependent cytosolicmalic enzyme 1 (ME1), NADP-dependent mitochondrial malic enzyme 3 (ME3),NAD(+)-dependent mitochondrial malic enzyme 2 (ME2), hematopoieticprostaglandin D2 synthase (PGDS), RAB32, RAB38, RAB7, cysteine stringprotein 2, gamma cysteine string protein, Beta cysteine string protein,F-box and leucine-rich repeat protein 11 (FBL11), JEMMA protein, PHDfinger protein 2, protein with GenBank Accession Number AAC83407, ABC50(TNF-alpha stimulated ABC protein), coronin 1B, coronin 1C,clipinE/coronin 6 type B, coronin 2A, coronin 2B, Sec61 alpha form 2,Sec61 alpha form 1, and vacuolar ATP synthase 21 kDa proteolipidsubunit), which include Drosophila and mammalian, preferably human,homologous polypeptides or proteins or sequences encoding thoseproteins.

Especially Preferred Embodiments are:

-   -   Drosophila Men (GadFly Accession Number CG10120), human        NADP-dependent cytosolic malic enzyme 1 (ME1; GenBank Accession        No. NM_(—)002395 for the cDNA, NP_(—)002386 for the protein), or        NADP-dependent mitochondrial malic enzyme 3 (ME3; GenBank        Accession No. NM_(—)006680 for the cDNA, NP_(—)006671 for the        protein), or NAD(+)-dependent mitochondrial malic enzyme 2 (ME2;        GenBank Accession No. NM_(—)002396.2 for the cDNA, NP_(—)002387        for the protein),    -   Drosophila Gst2 (GadFly Accession Number CG8938), human        hematopoietic prostaglandin D2 synthase (PGDS; GenBank Accession        No. NM_(—)014485 for the cDNA, NP_(—)055300 for the protein),        mouse hematopoietic prostaglandin D2 synthase 2 (Ptgds2; GenBank        Accession No. NM_(—)019455 for the cDNA; glutathione-requiring        prostaglandin D synthase), rat hematopoietic prostaglandin D2        synthase 2 (Ptgds2; GenBank Accession No. NM_(—)031644 for the        cDNA; glutathione-requiring prostaglandin D synthase),    -   Drosophila RabRP1 (GadFly Accession Number CG8024), human Rab32        (GenBank Accession No. NM_(—)006834 for the cDNA, NP_(—)006825        for the protein, formerly XM_(—)004076, human Rab38 (GenBank        Accession No. NM_(—)022337 for the cDNA, NP_(—)071732 for the        protein, formerly XM_(—)015771, mouse Rab32 (GenBank Accession        No. NM_(—)026405 for the cDNA), mouse Rab38 (GenBank Accession        No. NM_(—)028238 for the cDNA), human Rab7 (GenBank Accession        No. NM_(—)003929 for the cDNA, NP_(—)003920 for the protein),    -   Drosophila Csp (GadFly Accession Number CG6395), human Csp        (EnsEMBL accession number ENST00000217123 for the cDNA; GenBank        Accession Number CAC15495.1 for the protein), human cysteine        string protein 1 (GenBank Accession No. S70515 for the protein),        human gamma cysteine string protein (unnamed protein product;        GenBank Accession No. AK097736 for the cDNA, BAC05155 for the        protein), human Beta cysteine string protein (GenBank Accession        No. Q9UF47),    -   Drosophila F-box protein (GadFly Accession Number CG11033),        human F-box and leucine-rich repeat protein 11 (GenBank        Accession No. NM_(—)012308 for the cDNA, NP_(—)036440.1 for the        protein) human JEMMA protein (GenBank Accession No. CAD30700 for        the protein), PDH finger protein 2 (GenBank Accession Number        NM_(—)005392 for the cDNA, NP_(—)005383 for the protein), human        protein similar to several hypothetical proteins (GenBank        Accession No. AAC83407 for the protein),    -   Drosophila ABC50 (GadFly Accession Number CG1703), human        TNF-alpha stimulated ABC protein (GenBank Accession No. AF027302        for the cDNA, AAC70891 for the protein), rat ABC50 (GenBank        Accession No. AF293383 for the cDNA),    -   Drosophila coro (GadFly Accession Number CG9446), human        actin-binding protein coronin 1B (GenBank Accession No.        NM_(—)020441 for the cDNA, NP_(—)065174 for the protein,        formerly GenBank Accession No. BC006449), human actin-binding        protein coronin 1C (GenBank Accession No. NM_(—)014325 for the        cDNA, NP_(—)055140 for the protein; GenBank Accession No.        BC002342), human coronin homologue (GenBank Accession No. X89109        for the cDNA, CAA61482 for the protein), human clipinE/coronin 6        type B (see FIG. 27; Seq ID NO: 8), human Coronin 2A (GenBank        Accession No. Q92828 for the protein), human Coronin 2B (GenBank        Accession No. Q9UQ03 for the protein),    -   Drosophila sec61 alpha (GadFly Accession Number CG9539), human        Sec61 alpha form 2 protein (GenBank Accession No. NM_(—)018144        for the cDNA, NP_(—)060614 for the protein, formerly GenBank        Accession No. AF346603), human Sec61 alpha form 1 protein        (GenBank Accession No. NEU NM_(—)013336.2 for the cDNA,        NP_(—)037468 for the protein, formerly AF346602), mouse Sec61        alpha-2 protein (GenBank Accession No. AF222748), mouse Sec61        isoform 1 protein (GenBank Accession No. AF145253),    -   Drosophila VhaPPA1-1 (GadFly Accession Number CG7007), human        ATPase, H+transporting, lysosomal 21 kD (vacuolar protein pump)        protein (GenBank Accession No. NM_(—)004047 for the cDNA,        NP_(—)004038 for the protein), and mouse ATPase, H+transporting,        lysosomal 21 kD (vacuolar protein pump) protein (GenBank        Accession No. NM 033617 for the cDNA) as proteins of the        invention.

The present invention discloses proteins, which are regulating theenergy homeostasis and fat metabolism especially the metabolism andstorage of triglycerides, and polynucleotides, which identify and encodethe proteins disclosed in this invention. The invention also relates tovectors, host cells, antibodies, and recombinant methods for producingthe polypeptides and polynucleotides of the invention. The inventionalso relates to the use of these sequences and effector moleculesthereof in the diagnosis, study, prevention, and treatment of diseasesand disorders, for example, but not limited to, metabolic diseases suchas obesity as well as related disorders such as eating disorder,cachexia, diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancer,e.g. cancers of the reproductive organs, and sleep apnea.

The proteins of the invention and nucleic acid molecules coding thereforare obtainable from insect or vertebrate species, e.g. mammals or birds.Particularly preferred are homologous nucleic acids, particularlynucleic acids encoding a human homologous protein of the invention asdescribed above.

The invention particularly relates to a nucleic acid molecule encoding apolypeptide contributing to regulating the energy homeostasis and themetabolism of triglycerides, wherein said nucleic acid moleculecomprises

-   -   (a) the nucleotide sequence encoding a protein of the invention        and/or a sequence complementary thereto,    -   (b) a nucleotide sequence which hybridizes at 50° C. in a        solution containing 1×SSC and 0.1% SDS to a sequence of (a),    -   (c) a sequence corresponding to the sequences of (a) or (b)        within the degeneration of the genetic code,    -   (d) a sequence which encodes a polypeptide which is at least        85%, preferably at least 90%, more preferably at least 95%, more        preferably at least 98% and up to 99.6% identical to the amino        acid sequence of a protein of the invention,    -   (e) a sequence which differs from the nucleic acid molecule        of (a) to (d) by mutation and wherein said mutation causes an        alteration, deletion, duplication and/or premature stop in the        encoded polypeptide or    -   (f) a partial sequence of any of the nucleotide sequences of (a)        to (e) having a length of at least 15 bases, preferably at least        20 bases, more preferably at least 25 bases and most preferably        at least 50 bases.

The invention is based on the finding that the proteins of the inventionand the polynucleotides encoding these, are involved in the regulationof triglyceride storage and therefore energy homeostasis. The inventiondescribes the use of compositions comprising these polynucleotides,polypeptides or effectors thereof, e.g. antibodies, biologically activenucleic acids, such as antisense molecules, RNAi molecules or ribozymes,aptamers, peptides or low-molecular weight molecules or other receptorsof the polypeptides or polynucleotides for the diagnosis, study,prevention, or treatment of diseases and disorders related thereto,including metabolic diseases such as obesity as well as relateddisorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, and sleep apnea.

Accordingly, the present invention relates to genes with novel functionsin body-weight regulation, energy homeostasis, metabolism, and obesity.To find genes with novel functions in energy homeostasis, metabolism,and obesity, a functional genetic screen was performed with the modelorganism Drosophila melanogaster (Meigen). Drosophila melanogaster isone of the most intensively studied organisms in biology and serves as amodel system for the investigation of many developmental and cellularprocesses common to higher eukaryotes, including humans (see, forexample, Adams et al., Science 287: 2185-2195 (2000)). The success ofDrosophila melanogaster as a model organism is largely due to the powerof forward genetic screens to identify the genes that are involved in abiological process (see, Johnston Nat Rev Genet 3: 176-188 (2002);Rorth, Proc Natl Acad Sci U S A 93: 12418-12422 (1996)). One resourcefor screening was a proprietary Drosophila melanogaster stock collectionof EP-lines. The P-vector of this collection has Gal4-UAS-binding sitesfused to a basal promoter that can transcribe adjacent genomicDrosophila sequences upon binding of Gal4 to UAS-sites. This enables theEP-line collection for overexpression of endogenous flanking genesequences. In addition, without activation of the UAS-sites, integrationof the EP-element into the gene is likely to cause a reduction of geneactivity, and allows determining its function by evaluating theloss-of-function phenotype.

Triglycerides are the most efficient storage for energy in cells, andare significantly increased in obese patients. In this invention, wehave used a genetic screen to identify, that mutations of a geneencoding a protein of the invention or homologous genes cause changes inthe body weight which is reflected by a significant change in thetriglyceride levels. In order to isolate genes with a function in energyhomeostasis, several thousand proprietary and publicly availableEP-lines were tested for their triglyceride content after a prolongedfeeding period (illustrated in more detail in the EXAMPLES). Lines withsignificantly changed triglyceride content were selected as positivecandidates for further analysis.

In this invention, the content of triglycerides of a pool of flies withthe same genotype after feeding for six days was analyzed using atriglyceride assay, as, for example, but not for limiting the scope ofthe invention, is described in more detail below in the examplessection. The change of triglyceride content due to the loss of a genefunction suggests gene activities in energy homeostasis in a dosedependent manner that controls the amount of energy stored astriglycerides.

The results of the triglyceride content analysis are shown in FIGS. 1,5, 8, 12, 17, 22, 25, 31, and 33. We found that homozygousHD-EP(3)31178, HD-EP(3)37100, EP(2)0641, HD-EP(2)26782, EP(3)3141,HD-EP(3)31735, HD-EP(X)10216, HD-EP(2)26155, EP(2)2108, EP(2)2567, andEP(3)3504 flies have a higher triglyceride content than the controls(average triglyceride levels). Therefore, the very likely loss of a geneactivity in the gene loci, where the EP-vectors are integrated, isresponsible for changes in the metabolism of the energy storagetriglycerides, therefore representing in all cases an obese fly model.The increase of triglyceride content due to the loss of a gene functionsuggests gene activities in energy homeostasis in a dose dependentmanner that controls the amount of energy stored as triglycerides.

Nucleic acids encoding the proteins of the present invention wereidentified using a plasmid-rescue technique. Genomic DNA sequences wereisolated that are localised directly 3′ to the EP vectors (hereinHD-EP(3)31178, HD-EP(3)37100, EP(2)0641, HD-EP(2)26782, EP(3)3141,HD-EP(3)31735, HD-EP(X)10216, HD-EP(2)26155, EP(2)2108, EP(2)2567, orEP(3)3504) integration. Using those isolated genomic sequences publicdatabases like Berkeley Drosophila Genome Project (GadFly, see alsoFlyBase (1999) Nucleic Acids Research 27:85-88) were screened therebyconfirming the homozygous viable integration side of the vectors intothe transcription units of the genes FIGS. 2, 6, 9, 13, 18, 23, 26, 32,and 34 show the molecular organisation of these gene loci.

The present invention is further describing polypeptides comprising theamino acid sequences of the proteins of the invention. Based uponhomology, the proteins of the invention and each homologous protein orpeptide may share at least some activity. No functional data describedthe regulation of body weight control and related metabolic diseasessuch as obesity are available in the prior art for the genes of theinvention.

The proteins of the invention and homologous proteins and nucleic acidmolecules coding therefor are obtainable from insect or vertebratespecies, e.g. mammals or birds. Particularly preferred are nucleic acidsencoding the human homologs of the proteins of the invention. Thepresent invention is describing polypeptides comprising the amino acidsequences of the proteins of the invention. Comparisons (ClustalX 1.8analysis or ClustalW 1.82 analysis, see for example Thompson J. D. etal., (1994) Nucleic Acids Res. 22(22):4673-4680; Thompson J. D., (1997)Nucleic Acids Res 25(24):4876-4882; Higgins, D. G. et al., (1996)Methods Enzymol. 266:383-402) between the respective proteins ofdifferent species (human and Drosophila) were conducted. Based uponhomology, the Drosophila proteins of the invention and each homologousprotein or peptide may share at least some activity. No functional datadescribed the regulation of body weight control and related metabolicdiseases such as obesity are available in the prior art for the genesclaimed in this invention.

Further, we show that the mouse homologues of the genes encoding theproteins of the invention are regulated by fasting, by high fat diet, orby genetically induced obesity. Furthermore, the expression of the mousehomologues of Men, Rab32, Csp, ABC50, and vATPase is upregulated duringadipocyte differentiation in vitro, and the expression of the mousehomologue of F-box is downregulated during adipocyte differentiation invitro (see EXAMPLES).

The invention also encompasses polynucleotides that encode the proteinsof the invention and homologous proteins. Accordingly, any nucleic acidsequence, which encodes the amino acid sequences of the proteins of theinvention, can be used to generate recombinant molecules that expressthe proteins of the invention. In a particular embodiment, the inventionencompasses the polynucleotide comprising the nucleic acid sequenceencoding the Drosophila or human proteins of the invention. It will beappreciated by those skilled in the art that as a result of thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding the proteins of the invention, some bearing minimal homology tothe nucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequences of the naturally occurring proteins of theinvention, and all such variations are to be considered as beingspecifically disclosed. Although nucleotide sequences which encode theproteins of the invention and their variants are preferably capable ofhybridising to the nucleotide sequences of the naturally occurringproteins of the invention under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding the proteins of the invention or their derivatives possessing asubstantially different codon usage. Codons may be selected to increasethe rate at which expression of the peptide occurs in a particularprokaryotic or eukaryotic host in accordance with the frequency withwhich particular codons are utilised by the host. Other reasons forsubstantially altering the nucleotide sequence encoding the proteins ofthe invention and their derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequences. The invention alsoencompasses production of DNA sequences, or portions thereof, whichencode the proteins of the invention and their derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding the proteins of theinvention any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridising to the claimed nucleotide sequences, and inparticular, those of the polynucleotide encoding the proteins of theinvention under various conditions of stringency. Hybridisationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex or probe, as taught in Wahl, G. M. and S. L. Berger(1987: Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; MethodsEnzymol. 152:507-511), and may be used at a defined stringency.Preferably, hybridization under stringent conditions means that afterwashing for 1 h with 1×SSC and 0.1% SDS at 50° C., preferably at 55° C.,more preferably at 62° C. and most preferably at 68° C., particularlyfor 1 h in 0.2×SSC and 0.1% SDS at 50° C., preferably at 55° C., morepreferably at 62° C. and most preferably at 68° C., a positivehybridization signal is observed. Altered nucleic acid sequencesencoding the proteins of the invention which are encompassed by theinvention include deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same orfunctionally equivalent proteins of the invention.

The encoded proteins may also contain deletions, insertions, orsubstitutions of amino acid residues, which produce a silent change andresult in functionally equivalent proteins of the invention. Deliberateamino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological activity ofthe proteins of the invention is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding the proteins of the invention. As used herein, anallele or allelic sequence is an alternative form of the gene, which mayresult from at least one mutation in the nucleic acid sequence. Allelesmay result in altered mRNAs or polypeptides whose structures or functionmay or may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes, which give rise to alleles,are generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The nucleic acid sequences encoding the proteins of the invention may beextended utilising a partial nucleotide sequence and employing variousmethods known in the art to detect upstream sequences such as promotersand regulatory elements. For example, one method which may be employed,“restriction-site”. PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). Inverse PCR may also be used to amplify or extendsequences using divergent primers based on a known region (Triglia, T.et al. (1988) Nucleic Acids Res. 16:8186). Another method which may beused is capture PCR which involves PCR amplification of DNA fragmentsadjacent to a known sequence in human and yeast artificial chromosomeDNA (Lagerstrom, M. et al. (PCR Methods Applic. 1:111-119). Anothermethod which may be used to retrieve unknown sequences is that ofParker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

In order to express biologically active proteins of the invention, thenucleotide sequences encoding the proteins of the invention optionallyin the form of fusion proteins, may be inserted into appropriateexpression vectors, i.e., a vector, which contains the necessaryelements for the transcription and translation of the inserted codingsequence. Methods, which are well known to those skilled in the art, maybe used to construct expression vectors containing sequences encodingthe proteins of the invention and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques. synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook, J. et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y.

Regulatory elements include for example a promoter, an initiation codon,a stop codon, a mRNA stability regulatory element, and a polyadenylationsignal. Expression of a polynucleotide can be assured by (i)constitutive promoters such as the Cytomegalovirus (CMV)promoter/enhancer region, (ii) tissue specific promoters such as theinsulin promoter (see, Soria et al., 2000, Diabetes 49:157), SOX2 genepromotor (see Li et al., 1998, Curr. Biol. 8:971-4), Msi-1 promotor (seeSakakibara et al., 1997, J. Neuroscience 17:8300-8312), alpha-cardiamyosin heavy chain promotor or human atrial natriuretic factor promotor(Klug et al., 1996, J. clin. Invest 98:216-24; Wu et al., 1989, J. Biol.Chem. 264:6472-79) or (iii) inducible promoters such as the tetracyclineinducible system. Expression vectors can also contain a selection agentor marker gene that confers antibiotic resistance such as the neomycin,hygromycin or puromycin resistance genes. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook, J. et al.(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y. and Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y. In a furtherembodiment of the invention, natural, modified or recombinant nucleicacid sequences encoding the proteins of the invention and homologousproteins may be ligated to a heterologous sequence to encode a fusionprotein.

A variety of expression vector/host systems may be utilised to containand express sequences encoding the proteins of the invention or fusionproteins. These include, but are not limited to, micro-organisms such asbacteria transformed with recombinant bacteriophage, plasmid, or cosmidDNA expression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.baculovirus, adenovirus, adeno-associated virus, lentiverus,retrovirus); plant cell systems transformed with virus expressionvectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)or with bacterial expression vectors (e.g. Ti or PBR322 plasmids); oranimal cell systems.

The presence of polynucleotide sequences encoding the proteins of theinvention can be detected by DNA-DNA or DNA-RNA hybridisation oramplification using probes or portions or fragments of polynucleotidesencoding the proteins of the invention. Nucleic acid amplification basedassays involve the use of oligonucleotides or oligomers based on thesequences encoding the proteins of the invention to detect transformantscontaining DNA or RNA encoding the proteins of the invention. As usedherein “oligonucleotides” or “oligomers” refer to a nucleic acidsequence of at least about 10 nucleotides and as many as about 60nucleotides, preferably about 15 to 30 nucleotides, and more preferablyabout 20-25 nucleotides, which can be used as a probe or amplimer.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labelled hybridisation or PCR probesfor detecting sequences related to polynucleotides encoding the proteinsof the invention include oligo-labelling, nick translation,end-labelling or PCR amplification using a labelled nucleotide, orenzymatic synthesis. These procedures may be conducted using a varietyof commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).

The presence of proteins of the invention in a sample can be determinedby immunological methods or activity measurement. A variety of protocolsfor detecting and measuring the expression of proteins, using eitherpolyclonal or monoclonal antibodies specific for the protein or reagentsfor determining protein activity are known in the art. Examples includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on the protein is preferred, but a competitivebinding assay may be employed. These and other assays are described,among other places, in Hampton, R. et al. (1990; Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al.(1983; J. Exp. Med. 158:1211-1216).

Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, co-factors, inhibitors, magneticparticles, and the like.

The nucleic acids encoding the proteins of the invention can be used togenerate transgenic animal or site specific gene modifications in celllines. Transgenic animals may be made through homologous recombination,where the normal locus of the genes encoding the proteins of theinvention is altered. Alternatively, a nucleic acid construct israndomly integrated into the genome. Vectors for stable integrationinclude plasmids, retrovirusses and other animal virusses, YACs, and thelike. The modified cells or animal are useful in the study of thefunction and regulation of the proteins of the invention. For example, aseries of small deletions and/or substitutions may be made in the genesthat encode the proteins of the invention to determine the role ofparticular domains of the protein, functions in pancreaticdifferentiation, etc.

Specific constructs of interest include anti-sense molecules, which willblock the expression of the proteins of the invention, or expression ofdominant negative mutations. A detectable marker, such as for examplelac-Z, may be introduced in the locus of the genes of the invention,where upregulation of expression of the genes of the invention willresult in an easily detected change in phenotype.

One may also provide for expression of the genes of the invention orvariants thereof in cells or tissues where it is not normally expressedor at abnormal times of development. In addition, by providingexpression of the proteins of the invention in cells in which they arenot normally produced, one can induce changes in cell behavior.

DNA constructs for homologous recombination will comprise at leastportions of the genes of the invention with the desired geneticmodification, and will include regions of homology to the target locus.DNA constructs for random integration need not include regions ofhomology to mediate recombination. Conveniently, markers for positiveand/or negative selection are included. Methods for generating cellshaving targeted gene modifications through homologous recombination areknown in the art. For embryonic stem (ES) cells, an ES cell line may beemployed, or embryonic cells may be obtained freshly from a host, e.g.mouse, rat, guinea pig etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in presence of leukemia inhibitingfactor (LIF).

When ES or embryonic cells have been transformed, they may be used toproduce transgenic animals. After transformation, the cells are platedonto a feeder layer in an appropriate medium. Cells containing theconstruct may be detected by employing a selective medium. Aftersufficient time for colonies to grow, they are picked and analyzed forthe occurrence of homologous recombination or integration of theconstruct. Those colonies that are positive may then be used for embryomanipulation and blastocyst injection. Blastocysts are obtained from 4to 6; week old superovulated females. The ES cells are trypsinized, andthe modified cells are injected into the blastocoel of the blastocyst.After injection, the blastocysts are returned to each uterine horn ofpseudopregnant females. Females are then allowed to go to term and theresulting offspring screened for the construct. By providing for adifferent phenotype of the blastocyst and the genetically modifiedcells, chimeric progeny can be readily detected. The chimeric animalsare screened for the presence of the modified gene and males and femaleshaving the modification are mated to produce homozygous progeny. If thegene alterations cause lethality at some point in development, tissuesor organs can be maintained as allogenic or congenic grafts ortransplants, or in vitro culture. The transgenic animals may be anynon-human mammal, such as laboratory animal, domestic animals, etc. Thetransgenic animals may be used in functional studies, drug screening,etc.

Diagnostics and Therapeutics

The data disclosed in this invention show that the nucleic acids andproteins of the invention and effector molecules thereof are useful indiagnostic and therapeutic applications implicated, for example but notlimited to, in metabolic disorders such as obesity as well as relateddisorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, and sleep apnea. Hence, diagnostic and therapeuticuses for the nucleic acids and proteins of the invention and effectorsthereof are, for example but not limited to, the following: (i) proteintherapeutic, (ii) small molecule drug target, (iii) antibody target(therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv)diagnostic and/or prognostic marker, (v) gene therapy (genedelivery/gene ablation), (vi) research tools, and (vii) tissueregeneration in vitro and in vivo (regeneration for all these tissuesand cell types composing these tissues and cell types derived from thesetissues).

The nucleic acids and proteins of the invention and effectors thereofare useful in diagnostic and therapeutic applications implicated invarious applications as described below. For example, but not limitedto, cDNAs encoding the proteins of the invention and particularly theirhuman homologues may be useful in gene therapy, and the proteins of theinvention and particularly their human homologues may be useful whenadministered to a subject in need thereof. By way of non-limitingexample, the compositions of the present invention will have efficacyfor treatment of patients suffering from, for example, but not limitedto, in metabolic disorders as described above.

The nucleic acids encoding the proteins of the invention, or fragmentsthereof, may further be useful in diagnostic applications, wherein thepresence or amount of the nucleic acids or the proteins are to beassessed. These materials are further useful in the generation ofantibodies that bind immunospecifically to the substances of theinvention for use in therapeutic or diagnostic methods.

For example, in one aspect, antibodies which are specific for theproteins of the invention may be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express the proteins ofthe invention. The antibodies may be generated using methods that arewell known in the art. Such antibodies may include, but are not limitedto, polyclonal, monoclonal, chimerical, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralising antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunised by injectionwith the proteins of the invention any fragment or oligopeptide thereofwhich has immunogenic properties. Depending on the host species, variousadjuvants may be used to increase immunological response. It ispreferred that the peptides, fragments, or oligopeptides used to induceantibodies to the proteins of the invention have an amino acid sequenceconsisting of at least five amino acids, and more preferably at least 10amino acids.

Monoclonal antibodies to the proteins of the invention may be preparedusing any technique which provides for the production of antibodymolecules by continuous cell lines in culture. These include, but arenot limited to, the hybridoma technique, the human B-cell hybridomatechnique, and the EBV-hybridoma technique, (Kohler, G. et al. (1975)Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods81:31-42; Cote, R. J. et al. Proc. Natl. Acad. Sci. 80:2026-2030; Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to producethe proteins of the invention—and -specific single chain antibodies.Antibodies with related specificity, but of distinct idiotypiccomposition, may be generated by chain shuffling from randomcombinatorial immunoglobulin libraries (Burton, D. R. (1991) Proc. Natl.Acad. Sci. 88:11120-3). Antibodies may also be produced by inducing invivo production in the lymphocyte population or by screening recombinantimmunoglobulin libraries or panels of highly specific binding reagentsas disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl.Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments, which contain specific binding sites for theproteins of the invention, may also be generated. For example, suchfragments include, but are not limited to, the F(ab′)₂ fragments whichcan be produced by Pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofF(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse, W. D. et al. (1989)Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding and immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between the proteins of the invention and their specificantibodies. A two-site, monoclonal-based immunoassay utilisingmonoclonal antibodies reactive to two non-interfering epitopes of aprotein of the invention is preferred, but a competitive binding assaymay also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides or fragmentsthereof, or nucleic acid effector molecules such as antisense molecules,aptamers, RNAi molecules or ribozymes may be used for therapeuticpurposes. In one aspect, aptamers, i.e. nucleic acid molecules, whichare capable of binding to a protein of the, invention and modulating itsactivity may be generated by a screening and selection procedureinvolving the use of combinatorial nucleic acid libraries.

In a further aspect, antisense molecules to the polynucleotide encodingthe proteins of the invention may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding the proteins of the invention. Thus, antisensemolecules may be used to modulate the activity of the proteins of theinvention, or to achieve regulation of gene function. Such technology isnow well know in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding the proteins of the invention.Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods, which are well known to those skilled in the art,can be used to construct recombinant vectors, which will expressantisense molecules complementary to the polynucleotides of the genesencoding the proteins of the invention. These techniques are describedboth in Sambrook et al. (supra) and in Ausubel et al. (supra). Genesencoding the proteins of the invention can be turned off by transforminga cell or tissue with expression vectors which express high levels ofpolynucleotide or fragment thereof which encodes the proteins of theinvention. Such constructs may be used to introduce untranslatable senseor antisense sequences into a cell. Even in the absence of integrationinto the DNA, such vectors may continue to transcribe RNA moleculesuntil they are disabled by endogenous nucleases. Transient expressionmay last for a month or more with a non-replicating vector and evenlonger if appropriate replication elements are part of the vectorsystem.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, e.g. DNA, RNA, or nucleic acid analoguessuch as PNA, to the control regions of the gene encoding the proteins ofthe invention, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it cause inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In; Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The antisense molecules may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyse thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridisation of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage.Examples, which may be used, include engineered hammerhead motifribozyme molecules that can be specifically and efficiently catalyseendonucleolytic cleavage of sequences encoding the proteins of theinvention. Specific ribozyme cleavage sites within any potential RNAtarget are initially identified by scanning the target molecule forribozyme cleavage sites which include the following sequences: GUA, GUU,and GUC. Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridisation with complementary oligonucleotides usingribonuclease protection assays.

Nucleic acid effector molecules, e.g. antisense molecules and ribozymesof the invention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesising oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding the proteins of the invention. Such DNA sequences may beincorporated into a variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesise antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues. RNA molecules may be modified toincrease intracellular stability and half-life. Possible modificationsinclude, but are not limited to, the addition of flanking sequences atthe 5′ and/or 3′ ends of the molecule or the use of phosphorothioate or2′ O-methyl rather than phosphodiesterase linkages within the backboneof the molecule. This concept is inherent in the production of PNAs andcan be extended in all of these molecules by the inclusion ofnon-traditional bases such as inosine, queosine, and wybutosine, as wellas acetyl-, methyl-, thio-, and similarly modified forms of adenine,cytidine, guanine, thymine, and uridine which are not as easilyrecognised by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods, which are well known in the art. Any of thetherapeutic methods described above may be applied to any suitablesubject including, for example, mammals such as dogs, cats, cows,horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of the proteins of theinvention, antibodies to the proteins of the invention, mimetics,agonists, antagonists, or inhibitors of the proteins of the invention.The compositions may be administered alone or in combination with atleast one other agent, such as stabilising compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones. The pharmaceuticalcompositions utilised in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations which, can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.). Pharmaceutical compositions can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g. by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilising processes. Thepharmaceutical composition may be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulphuric,acetic, lactic, tartaric, malic, succinic, etc. After pharmaceuticalcompositions have been prepared, they can be placed in an appropriatecontainer and labelled for treatment of an indicated condition. Foradministration of the proteins of the invention, such labelling wouldinclude amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart. For any compounds, the therapeutically effective does can beestimated initially either in cell culture assays, e.g. of preadipocytecell lines, or in animal models, usually mice, rabbits, dogs, or pigs.The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. A therapeutically effective dose refers to that amount of activeingredient, for example the nucleic acids or proteins of the inventionor fragments thereof, or antibodies, which is sufficient for treating aspecific condition. Therapeutic efficacy and toxicity may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g. ED50 (the dose therapeutically effective in 50% of thepopulation) and LD50 (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD50/ED50. Pharmaceuticalcompositions, which exhibit large therapeutic indices, are preferred.The data obtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage from employed,sensitivity of the patient, and the route of administration. The exactdosage will be determined by the practitioner, in light of factorsrelated to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors, which may be takeninto account, include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation. Normal dosage amounts may vary from 0.1to 100,000 micrograms, up to a total dose of about 1 g, depending uponthe route of administration. Guidance as to particular dosages andmethods of delivery is provided in the literature and generallyavailable to practitioners in the art. Those skilled in the art employdifferent formulations for nucleotides than for proteins or theirinhibitors. Similarly, delivery of polynucleotides or polypeptides willbe specific to particular cells, conditions, locations, etc.

In another embodiment, antibodies which specifically bind the proteinsof the invention may be used for the diagnosis of conditions or diseasescharacterised by or associated with over- or underexpression of theproteins of the invention, or in assays to monitor patients beingtreated with the proteins of the invention, agonists, antagonists orinhibitors. The antibodies useful for diagnostic purposes may beprepared in the same manner as those described above for therapeutics.Diagnostic assays for the proteins of the invention include methods,which utilise the antibody and a label to detect the proteins of theinvention in human body fluids or extracts of cells or tissues. Theantibodies may be used with or without modification, and may be labelledby joining them, either covalently or non-covalently, with a reportermolecule. A wide variety of reporter molecules which are known in theart may be used several of which are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring theproteins of the invention are known in the art and provide a basis fordiagnosing altered or abnormal levels of expression of the proteins ofthe invention. Normal or standard values for expression of the proteinsof the invention are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibodies to the proteins of the invention under conditions suitablefor complex formation. The amount of standard complex formation may bequantified by various methods, but preferably by photometric means.Quantities of the proteins of the invention expressed in control anddisease samples, e.g. from biopsied tissues are compared with thestandard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides specific forthe proteins of the invention may be used for diagnostic purposes. Thepolynucleotides, which may be used, include oligonucleotide sequences,antisense RNA and DNA molecules, and PNAs. The polynucleotides may beused to detect and quantitate gene expression in biopsied tissues inwhich expression of the proteins of the invention may be correlated withdisease. The diagnostic assay may be used to distinguish betweenabsence, presence, and excess expression of the proteins of theinvention, and to monitor regulation of the levels of the proteins ofthe invention during therapeutic intervention.

In one aspect, hybridisation with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding the proteins of the invention and homologous proteins orclosely related molecules, may be used to identify nucleic acidsequences which encode the respective protein. The hybridisation probesof the subject invention may be DNA or RNA and are preferably derivedfrom the nucleotide sequences of the polynucleotides encoding theDrosophila or human proteins of the invention or from genomic sequenceincluding promoter, enhancer elements, and introns of the naturallyoccurring gene.

Hybridisation probes may be labelled by a variety of reporter groups,for example, radionuclides such as ³²P or ³⁵S, or enzymatic labels, suchas alkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences specific for the proteins of the invention andhomologous nucleic acids may be used for the diagnosis of conditions ordiseases, which are associated with expression of the proteins of theinvention. Examples of such conditions or diseases include, but are notlimited to, pancreatic diseases and disorders, including diabetes.Polynucleotide sequences specific for the proteins of the invention andhomologous proteins may also be used to monitor the progress of patientsreceiving treatment for pancreatic diseases and disorders, includingdiabetes. The polynucleotide sequences encoding the proteins of theinvention may be used in Southern or Northern analysis, dot blot, orother membrane-based technologies; in PCR technologies; or in dip stick,pin, ELISA or chip assays utilising fluids or tissues from patientbiopsies to detect altered gene expression.

In a particular aspect, the nucleotide sequences encoding the proteinsof the invention and homologous proteins may be useful in assays thatdetect activation or induction of various metabolic diseases such asobesity as well as related disorders such as eating disorder, cachexia,diabetes mellitus, hypertension; coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancersof the reproductive organs, and sleep apnea. The nucleotide sequencesencoding the proteins of the invention may be labelled by standardmethods, and added to a fluid or tissue sample from a patient underconditions suitable for the formation of hybridisation complexes. Aftera suitable incubation period, the sample is washed and the signal isquantitated and compared with a standard value. If the amount of signalin the biopsied or extracted sample is significantly altered from thatof a comparable have hybridised with nucleotide sequences in the sample,and the presence of altered levels of nucleotide sequences encoding theproteins of the invention in the sample indicates the presence of theassociated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of the proteins of the invention, a normal or standardprofile for expression is established. This may be accomplished bycombining body fluids or cell extracts taken from normal subjects,either animal or human, with sequences, or fragments thereof, whichencode the proteins of the invention, under conditions suitable forhybridisation or amplification. Standard hybridisation may be quantifiedby comparing the values obtained from normal subjects with those from anexperiment where a known amount of a substantially purifiedpolynucleotide is used. Standard values obtained from normal samples maybe compared with values obtained from samples from patients who aresymptomatic for disease. Deviation between standard and subject valuesis used to establish the presence of disease. Once disease isestablished and a treatment protocol is initiated, hybridisation assaysmay be repeated on a regular basis to evaluate whether the level ofexpression in the patient begins to approximate that, which is observedin the normal patient. The results obtained from successive assays maybe used to show the efficacy of treatment over a period ranging fromseveral days to months.

With respect to metabolic diseases such as described above, the presenceof an unusual amount of transcript in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the pancreatic diseases and disorders. Additionaldiagnostic uses for oligonucleotides designed from the sequencesencoding the proteins of the invention may involve the use of PCR. Sucholigomers may be chemically synthesised, generated enzymatically, orproduced from a recombinant source. Oligomers will preferably consist oftwo nucleotide sequences, one with sense orientation(5prime.fwdarw.3prime) and another with antisense (3prime.rarw.5prime),employed under optimised conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantification of closely related DNA orRNA sequences.

In another embodiment of the invention, the nucleic acid sequences,which encode the proteins of the invention, may also be used to generatehybridisation probes, which are useful for mapping the naturallyoccurring genomic sequence. The sequences may be mapped to a particularchromosome or to a specific region of the chromosome using well knowntechniques. Such techniques include FISH, FACS, or artificial chromosomeconstructions, such as yeast artificial chromosomes, bacterialartificial chromosomes, bacterial P1 constructions or single chromosomecDNA libraries as reviewed in Price, C. M. (1993) Blood Rev. 7:127-134,and Trask, B. J. (1991) Trends Genet 7:149-154. FISH (as described inVerma et al. (1988) Human Chromosomes: A Manual of Basic Techniques,Pergamon Press, New York, N.Y.) may be correlated with other physicalchromosome mapping techniques and genetic map data. Examples of geneticmap data can be found in the 1994 Genome Issue of Science (265:1981f).Correlation between the location of the genes encoding the proteins ofthe invention on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help to delimit the region ofDNA associated with that genetic disease.

The nucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier or affectedindividuals. An analysis of polymorphisms, e.g. single nucleotidepolymorphisms may be carried out. Further in situ hybridization ofchromosomal preparations and physical mapping techniques such as linkageanalysis using established chromosomal markers may be used for extendinggenetic maps. Often the placement of a gene on the chromosome of anothermammalian species, such as mouse, may reveal associated markers even ifthe number or arm of a particular human chromosome is not known. Newsequences can be assigned to chromosomal arms or parts thereof, byphysical mapping. This provides valuable information to investigatorssearching for disease genes using positional cloning or other genediscovery techniques. Once the disease or syndrome has been crudelylocalized by genetic linkage to a particular genomic region, forexample, AT to 11q22-23 (Gatti, R. A. et al. (1988) Nature 336:577-580),any sequences mapping to that area may represent associated orregulatory genes for further investigation. The nucleotide sequences ofthe subject invention may also be used to detect differences in thechromosomal location due to translocation, inversion, etc. among normal,carrier or affected individuals.

In another embodiment of the invention, the proteins of the invention,their catalytic or immunogenic fragments or oligopeptides thereof, an invitro model, a genetically altered cell or animal, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. One can identify effectors, e.g. receptors, enzymes,proteins, ligands, or substrates that bind to, modulate or mimic theaction of one or more of the proteins of the invention. The protein orfragment thereof employed in such screening may be free in solution,affixed to a solid support, borne on a cell surface, or locatedintracellularly. The formation, of binding complexes, between theproteins of the invention and the agent tested, may be measured. Agentscould also, either directly or indirectly, influence the activity of theproteins of the invention. Agents may also interfere withposttranslational modifications of the protein, such as phosphorylationand dephosphorylation, acetylation, alkylation, ubiquitination,proteolytic processing, subcellular localization, or degradation.Moreover, agents could influence the dimerization or oligomerization ofthe proteins of the invention or, in a heterologous manner, of theproteins of the invention with other proteins, for example, but notexclusively, ion channels, enzymes, receptors, or translation factors.Agents could also act on the physical interaction of the proteins ofthis invention with other proteins, which are required for proteinfunction, for example, but not exclusively, their downstream signalling.Methods for determining protein-protein Interaction are well known inthe art. For example binding of a fluorescently labeled peptide derivedfrom the interacting protein to the protein of the Invention, or viseversa, could be detected by a change in polarisation. In case that bothbinding partners, which can be either the full length proteins as wellas one binding partner as the full length protein and the other justrepresented as a peptide are fluorescently labeled, binding could bedetected by fluorescence energy transfer (FRET) from one fluorophore tothe other. In addition, a variety of commercially available assayprinciples suitable for detection of protein-protein interaction arewell known in the art, for example but not exclusively AlphaScreen(PerkinElmer) or Scintillation Proximity Assays (SPA) by Amersham.Alternatively, the interaction of the proteins of the invention withcellular proteins could be the basis for a cell-based screening assay,in which both proteins are fluorescently labeled and interaction of bothproteins is detected by analysing cotranslocation of both proteins witha cellular imaging reader, as has been developed for example, but notexclusively, by Cellomics or EvotecOAl. In all cases the two or morebinding partners can be different proteins with one being the protein ofthe invention, or in case of dimerization and/or oligomerization theprotein of the invention itself. Proteins of the invention, for whichone target mechanism of interest, but not the only one, would be suchprotein/protein interactions are CSP, F-box, coronin, ABC50, and Sec61alpha.

Assays for determining enzymatic activity of the proteins of theinvention are well known in the art. For example, but not exclusively,the activity of malic enzyme could be determined by monitoring theincrease of NADPH concentration during enzymatic reaction by the Beutlerassay (Beutler E. (1970) Br. J. Haematol. 18:117-121). GST2 activitycould for example be measured by spectrometric methods based onmonitoring prostaglandine synthetic activity or, glutathioneS-transferase activity (Pinzar et al. (2000) J. Biol. Chem.275:31239-31244). The GTPase activity of RabR1 and the ATPase activityof VhaPPA1-1 could represent target mechanisms for these enzymes.Examples for addressing posttranslational modification are thepalmitoylation and farnesylation of RabRP1 and CSP. In that case, theenzymes mediating such posttranslational modification would be targeted,an approach very well known in the art for the farnesylation of the Rasprotein (Prendergast G. C. and Rane N. (2001) Expert Opin Investig Drugs10(12):2105-2116).

Of particular interest are screening assays for agents that have a lowtoxicity for mammalian cells. The term “agent” as used herein describesany molecule, e.g. protein or pharmaceutical, with the capability ofaltering or mimicking the physiological function of one or more of theproteins of the invention. Candidate agents encompass numerous chemicalclasses, though typically they are organic molecules, preferably smallorganic compounds having a molecular weight of more than 50 and lessthan about 2,500 Daltons. Candidate agents comprise functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise carbocyclic orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups.

Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acidsand derivatives, structural analogs or combinations thereof. Candidateagents are obtained from a wide variety of sources including librariesof synthetic or natural compounds. For example, numerous means areavailable for random and directed synthesis of a wide variety of organiccompounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Where the screening assay is a binding assay, one ormore of the molecules may be joined to a label, where the label candirectly or indirectly provide a detectable signal.

Another technique for drug screening, which may be used, provides forhigh throughput screening of compounds having suitable binding affinityto the protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to the proteins of the inventionlarge numbers of different small test compounds, e.g. aptamers,peptides, low-molecular weight compounds etc., are provided orsynthesized on a solid substrate, such as plastic pins or some othersurface. The test compounds are reacted with the proteins or fragmentsthereof, and washed. Bound proteins are then detected by methods wellknown in the art. Purified proteins can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support. In another embodiment, onemay use competitive drug screening assays in which neutralizingantibodies capable of binding the protein specifically compete with atest compound for binding the protein. In this manner, the antibodiescan be used to detect the presence of any peptide, which shares one ormore antigenic determinants with the protein.

The nucleic acids encoding the proteins of the invention can be used togenerate transgenic cell lines and animals. These transgenic animals areuseful in the study of the function and regulation of the proteins ofthe invention in vivo. Transgenic animals, particularly mammaliantransgenic animals, can serve as a model system for the investigation ofmany developmental and cellular processes common to humans. Transgenicanimals may be made through homologous recombination in embryonic stemcells, where the normal locus of the gene encoding the protein of theinvention is mutated. Alternatively, a nucleic acid construct encodingthe protein is injected into oocytes and is randomly integrated into thegenome. One may also express the genes of the invention or variantsthereof in tissues where they are not normally expressed or at abnormaltimes of development. Furthermore, variants of the genes of theinvention like specific constructs expressing anti-sense molecules orexpression of dominant negative mutations, which will block or alter theexpression of the proteins of the invention may be randomly integratedinto the genome. A detectable marker, such as lac Z or luciferase may beintroduced into the locus of the genes of the invention, whereupregulation of expression of the genes of the invention will result inan easily detectable change in phenotype. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, yeastartificial chromosomes (YACs), and the like. DNA constructs forhomologous recombination will contain at least portions of the genes ofthe invention with the desired genetic modification, and will includeregions of homology to the target locus. Conveniently, markers forpositive and negative selection are included. DNA constructs for randomintegration do not need to contain regions of homology to mediaterecombination. DNA constructs for random integration will consist of thenucleic acids encoding the proteins of the invention, a regulatoryelement (promoter), an intron and a poly-adenylation signal. Methods forgenerating cells having targeted gene modifications through homologousrecombination are known in the field. For embryonic stem (ES) cells, anES cell line may be employed, or embryonic cells may be obtained freshlyfrom a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown onan appropriate fibroblast-feeder layer and are grown in the presence ofleukemia inhibiting factor (LIF). ES or embryonic cells may betransfected and can then be used to produce transgenic animals. Aftertransfection, the ES cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be selected byemploying a selection medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination. Colonies that are positive may then be used for embryomanipulation and morula aggregation. Briefly, morulae are obtained from4 to 6 week old superovulated females, the Zona Pellucida is removed andthe morulae are put into small depressions of a tissue culture dish. TheES cells are trypsinized, and the modified cells are placed into thedepression closely to the morulae. On the following day the aggregatesare transfered into the uterine horns of pseudopregnant females. Femalesare then allowed to go to term. Chimeric offsprings can be readilydetected by a change in coat color and are subsequently screened for thetransmission of the mutation into the next generation (F1-generation).Offspring of the F1-generation are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallogenic or congenic grafts or transplants, or in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimal, domestic animals, etc., for example, mouse, rat, guinea pig,sheep, cow, pig, and others. The transgenic animals may be used infunctional studies, drug screening, and other applications and areuseful in the study of the function and regulation of the proteins ofthe invention in vivo.

Finally, the invention also relates to a kit comprising at least one of

-   -   (a) a nucleic acid molecule encoding one of the proteins of the        invention or a fragment thereof;    -   (b) a vector comprising the nucleic acid of (a);    -   (c) a host cell comprising the nucleic acid of (a) or the vector        of (b);    -   (d) a polypeptide encoded by the nucleic acid of (a);    -   (e) a fusion polypeptide encoded by the nucleic acid of (a);    -   (f) an antibody, an aptamer or another receptor against the        nucleic acid of (a) or the polypeptide of (d) or (e) and    -   (g) an anti-sense oligonucleotide of the nucleic acid of (a).

The kit may be used for diagnostic or therapeutic purposes or forscreening applications as described above. The kit may further containuser instructions.

The Figures show:

FIG. 1 shows the increase of triglyceride content of HD-EP(3)31178 andHD-EP(3)37100 Drosophila Men mutant caused by homozygous viableintegration of the P-vector (in comparison to controls withoutintegration of this vector).

FIG. 2 shows the molecular organisation of the mutated Men protein genelocus.

FIG. 3A shows the comparison (CLUSTAL W 1.82 multiple sequencealignment) of Men proteins from different species, MEN1_Hs refers tohuman malic enzyme 1 (GenBank Accession No. NP_(—)002386), MEN3_Hsrefers to human malic enzyme 3 (GenBank Accession No. NP_(—)006671), andMEN_Dm refers to the protein encoded by Drosophila Men gene with GadFlyAccession No. CG10120. Gaps in the alignment are represented as -.

FIG. 3B shows the nucleic acid sequence of human malic enzyme 1 (SEQ IDNO: 1).

FIG. 3C shows the amino acid sequence (one-letter code) of human malicenzyme 1 (SEQ ID NO: 2).

FIG. 3D shows the nucleic acid sequence of human malic enzyme 3 (SEQ IDNO: 3).

FIG. 3E shows the amino acid sequence (one-letter code) of human malicenzyme 3 (SEQ ID NO: 4).

FIG. 3F shows the nucleic acid sequence of human malic enzyme 2 (SEQ IDNO: 42).

FIG. 3G shows the amino acid sequence (one-letter code) of human malicenzyme 2 (SEQ ID NO: 43).

FIG. 4 shows the expression of the Men gene in mammalian tissues.

FIG. 4A shows the real-time PCR analysis of Men in wildtype mousetissues.

FIG. 4B shows the real-time PCR mediated comparison of Men expressionduring differentiation of mammalian fibroblast (3T3-L1) cells frompre-adipocytes to mature adipocytes.

FIG. 5 shows increase of triglyceride content of EP(2)0641 Gst 2 mutantflies caused by homozygous or heterozygous viable integration of theP-vector (in comparison to controls without integration of this vector).

FIG. 6A shows the molecular organisation of the mutated GST2 gene locus.

FIG. 6B shows the nucleic acid sequence of human hematopoieticprostaglandin D2 synthase (PGDS) (SEQ ID NO: 5).

FIG. 6C shows the amino acid sequence (one-letter code) of humanhematopoietic prostaglandin D2 synthase (PGDS) (SEQ ID NO: 6).

FIG. 7 shows the expression of the Gst2 gene in mammalian tissues.

FIG. 7A shows the real-time PCR analysis of Gst2 expression in ob/obmice compared with wildtype mouse tissues (shown as fold expression ofGst2 in ob/ob versus wild type mice).

FIG. 7B shows the real-time PCR analysis of Gst2 expression in high fatdiet fed mice compared with wildtype mouse tissues (shown as foldexpression of Gst2 in ob/ob versus wild type mice).

FIG. 8 shows the increase of triglyceride content of HD-EP(2)26782Rab-RP1 mutant flies caused by homozygous viable or heterozygousintegration of the P-vector (in comparison to controls withoutintegration of this vector).

FIG. 9 shows the molecular organisation of the mutated Rab-RP1 genelocus.

FIG. 10 shows the comparison (CLUSTAL W 1.82 multiple sequencealignment) of Rab proteins from different species, CG8024_Dm refers tothe protein encoded by Drosophila Rab-RP1 gene with GadFly Accession No.CG8024, RAB32_Hs refers to human RAB32, member of RAS oncogene family(GenBank Accession No. NP_(—)006825), RAB38_Hs refers to human RAB38,Rab-related GTP-binding protein (GenBank Accession No. NP_(—)071732),and RAB7_Hs refers to human RAB7, member of RAS oncogene family-like 1(GenBank Accession No. NP_(—)003920). Gaps in the alignment arerepresented as -.

FIG. 11 shows the expression of the Rab32 and Rab38 genes in mammaliantissues.

FIG. 11A shows the real-time PCR analysis of Rab32 in wildtype mousetissues.

FIG. 11B shows the real-time PCR analysis of Rab38 in wildtype mousetissues.

FIG. 11C shows the real-time PCR mediated comparison of Rab32 expressionin different mouse models.

FIG. 11D shows the real-time PCR mediated comparison of Rab38 expressionin different mouse models.

FIG. 11E shows the real-time PCR mediated comparison of Rab32 expressionin genetically obese (db/db) and wildtype mice.

FIG. 11F shows the real-time PCR mediated comparison of Rab38 expressionin genetically obese (db/db) and wildtype mice.

FIG. 11G shows the real-time PCR mediated comparison of Rab32 expressionduring differentiation of mammalian fibroblast (3T3-L1) cells frompre-adipocytes to mature adipocytes.

FIG. 12 shows the increase of triglyceride content of EP(3)3141 CSPmutant flies caused by homozygous viable integration of the P-vector (incomparison to controls without integration of this vector).

FIG. 13 shows the molecular organisation of the mutated Csp gene locus.

FIG. 14 shows the cDNA sequence of the human Csp (SEQ ID NO: 7).

FIG. 15 shows the comparison (CLUSTAL W 1.82 multiple sequencealignment) of Csp proteins from different species, Beta-Csp_Hs refers tohuman Beta cysteine string protein (GenBank Accession No. Q9UF47),Csp_Hs refers to human cysteine string protein 2 (GenBank Accession No.S70516) CG6395_Dm refers to the protein encoded by Drosophila Csp genewith GadFly Accession No. CG6395, and Gamma-Csp_Hs refers to humanunnamed protein product (GenBank Accession No. BAC05155). Gaps in thealignment are represented as -.

FIG. 16 shows the expression of the Csp gene in mammalian tissues.

FIG. 16A shows the real-time PCR mediated comparison of Csp expressionduring differentiation of mammalian fibroblast (3T3-L1) cells frompre-adipocytes to mature adipocytes.

FIG. 16B shows the real-time PCR mediated comparison of Csp expressionduring differentiation of mammalian fibroblast (3T3-F442A) cells frompre-adipocytes to mature adipocytes.

FIG. 16C shows the real-time PCR mediated comparison of Csp expressionduring differentiation of mammalian TA1 cells from pre-adipocytes tomature adipocytes.

FIG. 17 shows the increase of the triglyceride content of HD-EP(3)31735F-box mutant flies caused by homozygous viable integration of theP-vector (in comparison to controls without integration of this vector).

FIG. 18 shows the molecular organisation of the mutated F-box proteinLilina/FBL7 gene locus.

FIG. 19 shows the Clustal X (1.81) multiple sequence alignment.NP_(—)036440 refers to the human F-box protein of the invention,chr12assembled refers to an assembled version of the F-box protein withhigh homologies to CG11033, mmBI653941_(—)3 refers to the mouse homolog,and CG11033 refers to the Drosophila F-box protein of the invention.

FIG. 20 shows the comparison (CLUSTAL W 1.82 multiple sequencealignment) of F-box proteins from different species, F-box_(—)11_Hsrefers to human F-box and leucine rich repeat protein 11 (GenBankAccession No. NP_(—)036440), JEMMA_Hs refers to human JEMMA protein(GenBank Accession No. CAD30700), CG11033_Dm refers to the proteinencoded by Drosophila gene with GadFly Accession No. CG11033,AAC83407_Hs refers to human protein similar to several hypotheticalproteins (GenBank Accession No. AAC83407), and PHD_finger_(—)2 refers tohuman PHD finger protein 2 (GenBank Accession No. NP_(—)005383). Gaps inthe alignment are represented as -.

FIG. 21 shows the expression of the F-box genes in mammalian tissues.

FIG. 21A shows the real-time PCR analysis of F-box in wildtype mousetissues.

FIG. 21B shows the real-time PCR mediated comparison of F-box expressionin genetically obese (db/db) and wildtype mice.

FIG. 21C shows the real-time PCR mediated comparison of F-box expressionin different mouse models.

FIG. 21D shows the real-time PCR mediated comparison of F-box expressionin wildtype mice hold under a high fat diet.

FIG. 21E shows the real-time PCR mediated comparison of F-box expressionduring differentiation of mammalian fibroblast (3T3-L1) cells frompre-adipocytes to mature adipocytes.

FIG. 21F shows the real-time PCR mediated comparison of F-box expressionduring differentiation of mammalian fibroblast (3T3-F442A) cells frompre-adipocytes to mature adipocytes.

FIG. 21G shows the real-time PCR mediated comparison of F-box expressionduring differentiation of mammalian TA1 cells from pre-adipocytes tomature adipocytes.

FIG. 22 shows the increase of triglyceride content of HD-EP(X)10216ABC50 mutant flies caused by homozygous viable integration of theP-vector (in comparison to controls without integration of this vector).

FIG. 23 shows the molecular organisation of the mutated ABC50 genelocus.

FIG. 24 shows the expression of the ABC50 gene in mammalian tissues.

FIG. 24A shows the real-time PCR analysis of ABC50 in wildtype mousetissues.

FIG. 24B shows the real-time PCR mediated comparison of ABC50 expressionduring differentiation of mammalian fibroblast (3T3-L1) cells frompre-adipocytes to mature adipocytes.

FIG. 24C shows the real-time PCR mediated comparison of ABC50 expressionduring differentiation of mammalian fibroblast (3T3-F442A) cells frompre-adipocytes to mature adipocytes.

FIG. 24D shows the real-time PCR mediated comparison of ABC50 expressionduring differentiation of mammalian TA1 cells from pre-adipocytes tomature adipocytes.

FIG. 25 shows the increase of triglyceride content of HD-EP(2)26155coronin mutant flies caused by homozygous or heterozygous viableintegration of the P-vector (in comparison to controls withoutintegration of this vector).

FIG. 26 shows the molecular organisation of the mutated coronin genelocus.

FIG. 27 shows the protein sequence (one-letter code) for human clipin E(SEQ ID NO: 8), which was reconstructed from human sequence NT010808 andmouse sequence BAB64362 using the program genewise

FIG. 28 shows the Clustal X (1.81) multiple sequence alignment of theamino acid sequences (one-letter code) for human coronin 1B (hs1B;GenBank Accession No. NP065174), human coronin 1C (hs1C; GenBankAccession No. NP055140), human clipinE (hs-clipin-gehewise; Seq ID NO:8), and Drosophila coronin (CG9446; GadFly Accession Number CG9446). Theidentities are 53-54% and the similarities 68-70% between the humancoronin proteins and the Drosophila protein.

FIG. 29 shows the comparison (CLUSTAL W 1.82 multiple sequencealignment) of coronin proteins from different species, Coronin_(—)1Crefers to human coronin 1C (GenBank Accession No. NP_(—)055140),Coronin_(—)1B refers to human coronin 1B (GenBank Accession No.NP_(—)065174), ClipinE_Hs refers to human clipin E (Seq ID NO: 8),Coronin_Hs refers to human coronin homologue (GenBank Accession No.CAA61482), CG9446_Dm refers to the protein encoded by Drosophila corogene with GadFly Accession No. CG9446, Coronin_(—)2B refers to humanCoronin 2B (GenBank Accession No. Q9UQ03), and Coronin_(—)2A refers tohuman Coronin 2A (GenBank Accession No. Q92828). Gaps in the alignmentare represented as -.

FIG. 30 shows the expression of the coronin 1B, Coronin1C, and Coronin6genes in mammalian tissues.

FIG. 30A shows the real-time PCR analysis of Coronin1B in wildtype mousetissues.

FIG. 30B shows the real-time PCR analysis of Coronin1C in wildtype mousetissues.

FIG. 30C shows the real-time PCR analysis of Coronin6 in wildtype mousetissues.

FIG. 30D shows the real-time PCR mediated comparison of Coronin 1Cexpression in different mouse models.

FIG. 31 shows the increase of triglyceride content of EP(2)2108 andEP(2)2567 Sec61 alpha mutant flies caused by heterozygous integration ofthe P-vector (in comparison to controls without integration of thisvectors).

FIG. 32 shows the molecular organisation of the mutated Sec61 alpha genelocus.

FIG. 33 shows the increase of triglyceride content of EP(3)3504VhaPPA1-1 mutant flies caused by homozygous viable or heterozygousintegration of the P-vector (in comparison to controls withoutintegration of this vector).

FIG. 34 shows the molecular organisation of the mutated VhaPPA1-1 genelocus.

FIG. 35 shows the transmembrane domain plot of VhaPPA1-1.

FIG. 36 shows the expression of the vATPase gene in mammalian tissues.

FIG. 36A shows the real-time PCR mediated comparison of vATPaseexpression during differentiation of mammalian fibroblast (3T3-L1) cellsfrom pre-adipocytes to mature adipocytes.

FIG. 36B shows the real-time PCR mediated comparison of vATPaseexpression during differentiation of mammalian fibroblast (3T3-F442A)cells from pre-adipocytes to mature adipocytes.

FIG. 36C shows the real-time PCR mediated comparison of vATPaseexpression during differentiation of mammalian TA1 cells frompre-adipocytes to mature adipocytes.

The examples illustrate the invention:

EXAMPLE 1 Measurement of Triglyceride Content in Drosophila

The change of triglyceride content of Drosophila melanogaster containinga special expression system (EP-element; Rorth P., Proc Natl Acad SciUSA 1996, 93(22):12418-12422) was measured. Mutant flies are obtainedfrom fly mutation stock collections (proprietary fly mutation stockcollection; P Insertion Mutation Stock Center, Sezged, Hungary). Theflies are grown under standard conditions known to those skilled in theart. In the course of the experiment, additional feedings with bakersyeast (Saccharomyces cerevisiae) are provided. The average increase oftriglyceride content of Drosophila containing the HD-EP(3)31178,HD-EP(3)37100, EP(2)0641, HD-EP(2)26782, EP(3)3141, HD-EP(3) 31735,HD-EP(X)10216, HD-EP(2)26155, EP(2)2108, EP(2)2567, and EP(3)3504vectors in homozygous or heterozygous integration was investigated incomparison to control flies (FIG. 1, 5, 8, 12, 17, 22, 25, 31, and 33).For determination of triglyceride, flies were incubated for 5 min at 90°C. in an aqueous buffer using a waterbath, followed by hot extraction.After another 5 min incubation at 90° C. and mild centrifugation, thetriglyceride content of the flies extract was determined using SigmaTriglyceride (INT 336-10 or -20) assay by measuring changes in theoptical density according to the manufacturer's protocol. As a referenceprotein content of the same extract was measured using BIO-RAD DCProtein Assay according to the manufacturer's protocol. The assays wererepeated three times.

The average triglyceride level of all male flies of the EP collection(referred to as ‘EP-control males’) is shown as 100% in FIG. 1, 5, 8,12, 17, 25, 31, and 33. The average triglyceride level of all femaleflies of the EP collection (referred to as ‘EP-control females’) isshown as 100% in FIG. 22.

Men

HD-EP(3)31178 and HD-EP(3)37100 homozygous flies show constantly ahigher triglyceride content than the controls (approx. 35-60%; columns 2and 3 in FIG. 1. Therefore, the loss of gene activity in the locus87C9-87D1 on chromosome 3R where the EP-vector of HD-EP(3)31178 andHD-EP(3)37100 flies is homozygous viably integrated, is responsible forchanges in the metabolism of the energy storage triglycerides, thereforerepresenting in both cases a model for obese flies.

Gst2

EP(2)0641 homozygous flies (obtained from the P Insertion Mutation StockCenter, Sezged, Hungary) show constantly a higher triglyceride contentthan the controls (approx. 70%; column 2 in FIG. 5). Therefore, the lossof gene activity in the locus 53F11 on chromosome 2R where the EP-vectorof EP(2)0641 flies is homozygous viably integrated, is responsible forchanges in the metabolism of the energy storage triglycerides, thereforerepresenting a model for obese flies. Even heterozygous integration ofEP(2)0641 causes an increase of about 40% of the triglyceride content inflies (see column 3 in FIG. 5).

RabRP1

HD-EP(2)26782 homozygous flies show constantly a higher triglyceridecontent than the controls (approx. 75%; column 2 in FIG. 8). Therefore,the loss of gene activity in the locus 45B3-45B4 on chromosome 2R wherethe EP-vector of HD-EP(2)26782 flies is homozygous viably integrated, isresponsible for changes in the metabolism of the energy storagetriglycerides, therefore representing a model for obese flies. Evenheterozygous integration of HD-EP(2)26782 causes an increase of about60% of the triglyceride content in flies (see column 3 in FIG. 8).

Csp

EP(3)3141 homozygous flies (obtained from the P Insertion Mutation StockCenter, Sezged, Hungary) show constantly a higher triglyceride contentthan the controls (approx. 65%; column 2 in FIG. 12). Therefore, theloss of gene activity in the locus 79E1-2 on chromosome 3L where theEP-vector of EP(3)3141 flies is homozygous viably integrated, isresponsible for changes in the metabolism of the energy storagetriglycerides, therefore representing a model for obese flies.

F-Box

HD-EP(3)31735 homozygous flies show constantly a higher triglyceridecontent than the controls (approx. 140%; column 2 in FIG. 17).Therefore, the loss of gene activity in the locus 85C6-7 on chromosome3R where the EP-vector of HD-EP(3)31735 flies is homozygous viablyintegrated, is responsible for changes in the metabolism of the energystorage triglycerides, therefore representing a model for obese flies.

ABC50

HD-EP(X)10216 homozygous flies show constantly a higher triglyceridecontent than the controls (approx. 130%; column 2 in FIG. 22).Therefore, the loss of gene activity in the locus 10C7 on chromosome Xwhere the EP-vector of HD-EP(X)10216 flies is homozygous viablyintegrated, is responsible for changes in the metabolism of the energystorage triglycerides, therefore representing a model for obese flies.

Coronin

HD-EP(2)26155 homozygous flies show constantly a higher triglyceridecontent than the controls (approx. 125%; column 2 in FIG. 25), and evenheterozygous flies show a higher triglyceride content than the controls(approx. 65%; column 3 in FIG. 25). Therefore, the loss of gene activityin the locus 42C8 on chromosome 2R where the EP-vector of HD-EP(2)26155flies is homozygous viably or heterozygous integrated, is responsiblefor changes in the metabolism of the energy storage triglycerides,therefore representing a model for obese flies.

Sec61alpha

EP(2)2108 and EP(2)2567 heterozygous flies (obtained from the PInsertion Mutation Stock Center, Sezged, Hungary) show constantly ahigher triglyceride content than the controls (approx. 75%; column 2 inFIG. 31 (‘EP(2)2108/CyO’); approx. 40% column 3 in FIG.31(‘EP(2)2567/CyO’). Therefore, the loss of gene activity in the locus26D6 on chromosome 2L where the EP-vector of EP(2)2108 and EP(2)2567flies are heterozygous integrated, is responsible for changes in themetabolism of the energy storage triglycerides, therefore representingin both cases a model for obese flies.

VATPase

EP(3)3504 homozygous flies (obtained from the P Insertion Mutation StockCenter, Sezged, Hungary) show constantly a higher triglyceride contentthan the controls (approx. 185%; column 2 in FIG. 33). Therefore, theloss of gene activity in the locus 88D8 on chromosome 3R where theEP-vector of EP(3)3504 flies is homozygous viably integrated, isresponsible for changes in the metabolism of the energy storagetriglycerides, therefore representing a model for obese flies. Evenheterozygous integration of EP(3)3504 causes an increase of about 40% ofthe triglyceride content in flies (see column 3 in FIG. 33).

EXAMPLE 2 Identification of Drosophila Genes Responsible for the Changesin the Metabolism of the Energy Storage Triglycerides

Using plasmid rescue method, genomic DNA sequences were isolated thatare localized directly adjacent in 3prime direction of the integrationsite of the EP vectors (herein HD-EP(3)31178, HD-EP(3)37100, EP(2)0641,HD-EP(2)26782, EP(3)3141, HD-EP(3)31735, HD-EP(X)10216, HD-EP(2)26155,EP(2)2108, EP(2)2567, and EP(3)3504). Using those isolated genomicsequences, public DNA sequence databases like Berkeley Drosophila GenomeProject (GadFly) were screened thereby identifying the integration sitesof the vectors. FIGS. 2, 6, 9, 13, 18, 23, 26, 32, and 34 show themolecular organization of these gene loci.

Men

In FIG. 2, genomic DNA sequence is represented by the assembly as adotted black line (from position 8468390 to 8480890 on chromosome 3R)that includes the integration sites of vector for line HD-EP(3)31178 andHD-EP(3)37100. Transcribed DNA sequences (expressed sequence tags, ESTs)and predicted exons are shown as bars in the lower two lines. Predictedexons of the cDNA with GadFly Accession Number CG10120 are shown as darkgrey bars and introns as light grey bars. Men protein encodes for a genethat is predicted by GadFly sequence analysis programs as AccessionNumber CG10120. Public DNA sequence databases (for example, NCBIGenBank) were screened thereby identifying the integration sites oflines HD-EP(3)31178 and HD-EP(3)37100, causing an increase oftriglyceride content. HD-EP(3)31178 and HD-EP(3)37100 is integrated intothe second exon in sense orientation of the cDNA with GadFly AccessionNumber CG10120. Therefore, expression of the cDNA encoding Men (GadFlyAccession Number CG10120) could be effected by homozygous integration ofvectors of line HD-EP(3)31178 or HD-EP(3)37100, leading to increase ofthe energy storage triglycerides.

Gst2

In FIG. 6, genomic DNA sequence is represented by the assembly as adotted black line (from position 1205500 to 12061250 on chromosome 2R)that includes the integration sites of vector for lines EP(2)0641.Transcribed DNA sequences (ESTs) and predicted exons are shown as barsin the lower two lines. Predicted exons of the cDNA with GadFlyAccession Number CG8938 are shown as dark grey bars and introns as lightgrey bars. GST2 encodes for a gene that is predicted by GadFly sequenceanalysis programs as Accession Number CG8938. Public DNA sequencedatabases (for example, NCBI GenBank) were screened thereby identifyingthe integration site of line EP(2)0641, causing an increase oftriglyceride content. EP(2)0641 is integrated 125 base pairs 5prime ofthe cDNA with Accession Number CG8938, encoding GST2 in senseorientation. Therefore, expression of the cDNA encoding Accession NumberCG8938 could be effected by homozygous integration of vectors of lineEP(2)0641, leading to increase of the energy storage triglycerides.

RabRP1

In FIG. 9, genomic DNA sequence is represented by the assembly as adotted black line (from position 4210418 to 4235418 on chromosome 2R)that includes the integration site of vector for line HD-EP(2)26782.Transcribed DNA sequences (ESTs) and predicted exons are shown as barsin the lower two lines. Predicted exons of the cDNA with GadFlyAccession Number CG8024 are shown as dark grey bars and introns as lightgrey bars. Rab-RP1 encodes for a gene that is predicted by GadFlysequence analysis programs as Accession Number CG8024. Public DNAsequence databases (for example, NCBI GenBank) were screened therebyidentifying the integration site of line HD-EP(2)26782, causing anincrease of triglyceride content. HD-EP(2)26782 is integrated in thecDNA at approximately 20 base pairs in antisense orientation of GadFlyAccession Number CG8024, encoding Rab-RP1. Therefore, expression of thecDNA encoding GadFly Accession Number CG8024 could be effected byhomozygous or heterozygous integration of vectors of line HD-EP(2)26782,leading to increase of the energy storage triglycerides.

Csp

In FIG. 13, genomic DNA sequence is represented by the assembly as adotted black line (from position 22101652 to 22114152 on chromosome 3L)that includes the integration site of vector for line EP(3)3141.Transcribed DNA sequences (ESTs) and predicted exons are shown as barsin the upper two lines. Predicted exons of the cDNA with GadFlyAccession Number CG6395 are shown as dark grey bars and introns as lightgrey bars. Csp encodes for a gene that is predicted by GadFly sequenceanalysis programs as Accession Number CG6395. Public DNA sequencedatabases (for example, NCBI GenBank) were screened thereby identifyingthe integration site of line EP(3)3141, causing an increase oftriglyceride content. EP(3)3141 is integrated into the cDNA at thesecond intron in antisense orientation of Accession Number CG6395,encoding cysteine string protein Csp. Therefore, expression of the cDNAencoding Accession Number CG6395 could be effected by homozygousintegration of vectors of lines EP(3)3141, leading to increase of theenergy storage triglycerides.

F-box

In FIG. 18, genomic DNA sequence is represented by the assembly as adotted black line (from position 4858500 to 4871000 on chromosome 3R)that includes the integration site of vector for line HD-EP(3)31735.Transcribed DNA sequences (ESTs) and predicted exons are shown as barsin the upper two lines. Predicted exons of the cDNA with GadFlyAccession Number CG11033 are shown as dark grey bars and introns aslight grey bars. F-box protein Lilina/FBL7 encodes for a gene that ispredicted by GadFly sequence analysis programs as Accession NumberCG11033. Public DNA sequence databases (for example, NCBI GenBank) werescreened thereby identifying the integration site of line HD-EP(3)31735,causing an increase of triglyceride content. HD-EP(3)31735 is integratedinto the promoter of in the 5prime in antisense orientation of the cDNAwith Accession Number CG11033. Therefore, expression of the cDNAencoding Accession Number CG11033 could be effected by homozygousintegration of vectors of line HD-EP(3)31735, leading to increase of theenergy storage triglycerides.

ABC50

In FIG. 23, genomic DNA sequence is represented by the assembly as adotted black line (from position 11379740 to 11404740 on chromosome X)that includes the integration site of vector for line HD-EP(X)10216.Transcribed DNA sequences (ESTs) and predicted exons are shown as barsin the upper two lines. Predicted exons of the cDNA with GadFlyAccession Number CG1703 are shown as dark grey bars and introns as lightgrey bars. ABC50 encodes for a gene that is predicted by GadFly sequenceanalysis programs as Accession Number CG1703. Public DNA sequencedatabases (for example, NCBI GenBank) were screened thereby identifyingthe integration sites of lines HD-EP(X)10216, causing an increase oftriglyceride content. HD-EP(X)10216 is integrated 45 base pairs 5primein antisense orientation of the cDNA with Accession Number CG1703.Therefore, expression of the cDNA encoding Accession Number CG1703 couldbe effected by homozygous integration of vectors of line HD-EP(X) 10216,leading to increase of the energy storage triglycerides.

Coronin

In FIG. 26, genomic DNA sequence is represented by the assembly as adotted black line (from position 1903188 to 1928188 on chromosome 2R)that includes the integration site of vector for line HD-EP(2)26155.Transcribed DNA sequences (ESTs) and predicted exons are shown as barsin the lower two lines. Predicted exons of the cDNA with GadFlyAccession Number CG9446 are shown as dark grey bars and introns as lightgrey bars coronin encodes for a gene that is predicted by GadFlysequence analysis programs as Accession Number CG9446. Public DNAsequence databases (for example, NCBI GenBank) were screened therebyidentifying the integration sites of lines HD-EP(2)26155, causing anincrease of triglyceride content. HD-EP(2)26155 is integrated at aboutbase pair 50 of the cDNA with Accession Number CG9446, encoding coroninin sense orientation. Therefore, expression of the cDNA encodingAccession Number CG9446 could be effected by homozygous integration ofvectors of lines HD-EP(2)26155, leading to increase of the energystorage triglycerides.

Sec61alpha

In FIG. 32, genomic DNA sequence is represented by the assembly as adotted black line (from position 6377343 to 6380768 on chromosome 2L)that includes the integration sites of vector for lines EP(2)2108 andEP(2)2567. Transcribed DNA sequences (ESTs) and predicted exons areshown as bars in the lower two lines. Predicted exons of the cDNA withGadFly Accession Number CG9539 are shown as dark grey bars and intronsas light grey bars. Sec61 alpha encodes for a gene that is predicted byGadFly sequence analysis programs as Accession Number CG9539. Public DNAsequence databases (for example, NCBI GenBank) were screened therebyidentifying the integration sites of lines EP(2)2108 and EP(2)2567,causing an increase of triglyceride content. EP(2)2108 and EP(2)2567 areboth integrated in the first intron of the cDNA with Accession NumberCG9539, encoding Sec61 alpha. Therefore, expression of the cDNA encodingAccession Number CG9539 could be effected by heterozygous integration ofvectors of lines EP(2)2108 and EP(2)2567, leading to increase of theenergy storage triglycerides.

vATPase

In FIG. 34, genomic DNA sequence is represented by the assembly as adotted black line (from position 10658674 to 10661799 on chromosome 3R)that includes the integration site of vector for line EP(3)3504.Transcribed DNA sequences (ESTs) and predicted exons are shown as barsin the lower two lines. Predicted exons of the cDNA with GadFlyAccession Number CG7007 are shown as dark grey bars and introns as lightgrey bars. VhaPPA1-1 encodes for a gene that is predicted by GadFlysequence analysis programs as Accession Number CG7007. Public DNAsequence databases (for example, NCBI GenBank) were screened therebyidentifying the integration site of line EP(3)3504, causing an increaseof triglyceride content. EP(3)3504 is integrated into the first exon inantisense orientation of the cDNA with Accession Number CG7007.Therefore, expression of the cDNA encoding Accession Number CG7007 couldbe effected by homozygous integration of vectors of line EP(3)3504,leading to increase of the energy storage triglycerides.

EXAMPLE 3 Identification of Mammalian Men, GST2, Rab-RP1, Csp, F-BoxLilina/FBL7, ABC50, Coronin, Sec61 Alpha, or VhaPPA1-1 Protein and GeneHomologs

The proteins of the invention and homologous proteins and nucleic acidmolecules coding therefore are obtainable from insect or vertebratespecies, e.g. mammals or birds. Particularly preferred are nucleic acidsencoding the Drosophila or human homologs of the proteins of theinvention. Sequences homologous to Drosophila proteins of the inventionwere identified using the publicly available program BLASTP 2.2.3 of thenon-redundant protein data base of the National Center for BiotechnologyInformation (NCBI) (see, Altschul et al., 1997, Nucleic Acids Res.25:3389-3402).

Men

Drosophila Men protein is in 545 amino acids 58% identical and 73%similar to human cytosolic malic enzyme 1 (ME1), NADP(+)-dependent(GenBank Accession Number NM_(—)002395 for the cDNA, NP_(—)002386 forthe protein), localized on chromosome 6. Drosophila Men protein is over537 amino acids 56% identical and 73% similar to human mitochondrialmalic enzyme 3, NADP(+)-dependent, pyruvic-malic carboxylase, malatedehydrogenasae, NADP-ME (GenBank Accession Number NM_(—)006680 for thecDNA, NP_(—)006671 for the protein), localized on chromosome 11.Drosophila Men protein is also homologous to human mitochondrial malicenzyme 2, NAD(+)-dependent (GenBank Accession No. NM_(—)002396.2 for thecDNA, NP_(—)002387 for the protein). An alignment of MEN from differentspecies has been done by the ClustaW program (see also FIG. 3A).

Gst2

Particularly preferred are human GST2 homologous nucleic acids,particularly nucleic acids encoding a human hematopoietic prostaglandinD2 synthase (glutathione-requiring prostaglandin D synthase, PGDS;GenBank Accession No. NM_(—)014485 for the cDNA, NP_(—)055300 for theprotein), mouse hematopoietic prostaglandin D2 synthase 2 (Ptgds2;GenBank Accession No. NM_(—)019455 for the cDNA), and rat hematopoieticprostaglandin D2 synthase 2 (Ptgds2; GenBank Accession No. NM_(—)31644for the cDNA). An alignment of GST2 from different species has been doneby the ClustaW program.

RabRP

Particularly preferred are human Rab-RP1 homologous nucleic acids,particularly nucleic acids encoding a human Rab32 (GenBank Accession No.NM_(—)006834 for the cDNA, NP_(—)006825 for the protein, formerlyXM_(—)004076, human Rab38 (GenBank Accession No. NM_(—)022337 for thecDNA, NP_(—)071732 for the protein, formerly XM_(—)015771, and humanRab7 (GenBank Accession No. NM_(—)003929 for the cDNA, NP_(—)003920 forthe protein). Drosophila Gene CG8024 shows 67% identity and 78%similiarity to human Rab32 in 209 amino acids, and Drosophila GeneCG8024 shows 72% identity and 83% similiarity to human Rab38 in 176amino acids. An alignment of Rab-RP1 from Drosophila and human has beendone by the ClustaW program (see also FIG. 10). NEU Drosophila RabRP1also shows homology to mouse Rab32 (GenBank Accession No. NM_(—)026405for the cDNA) and mouse Rab38 (GenBank Accession No. NM_(—)028238 forthe cDNA).

Csp

Particularly preferred are human Csp homologous nucleic acids,particularly nucleic acids encoding human Csp (EnsEMBL accession numberENST00000217123 for the cDNA; GenBank Accession Number CAC15495.1 forthe protein; see FIG. 14, SEQ ID NO: 7), human cysteine string protein 1(GenBank Accession No. S70515 for the protein), human gamma cysteinestring protein (unnamed protein product; GenBank Accession No. AK097736for the cDNA, BAC05155 for the protein), human Beta cysteine stringprotein (GenBank Accession No. Q9UF47). The cDNA shown in FIG. 14 wasgenerated from the genomic sequence AL118506 (located on human chromosom20) by applying the Genscan program. Drosophila Gene CG6395 shows 61%identity and 73% similiarity to human cysteine string protein (AccessionNumber CAC15495.1) in 165 amino acids (amino acids 8 to 165 in CG6395).The highest similiarity is found in the conserved DNAJ—domain and thecys-string of these proteins, with 77% identity and 88% similiarity inthe DNAJ domain. An alignment of Csp from different species has beendone by the ClustaW program.

F-Box

Particularly preferred are nucleic acids encoding Drosophila F-boxprotein Lilina/FBL7 (GadFly Accession Number CG11033), human F-boxprotein Lilina/FBL7 (similar to human F-box and leucine-rich repeatprotein 11; GenBank Accession No. NM_(—)012308 for the cDNA andNP_(—)036440.1 for the protein), human JEMMA protein (GenBank AccessionNo. CAD30700 for the protein), NEU PDH finger protein 2 (GenBankAccession Number NM_(—)005392 for the cDNA, NP_(—)005383 for theprotein), NEU human protein similar to several hypothetical proteins(GenBank Accession No. AAC83407 for the protein). An alignment of F-boxprotein Lilina/FBL7 from different species has been done by the ClustaLX (1.8.) multiple sequence alignment program (see FIG. 19) and Clustal W(1.82) multiple sequence alignment program (see FIG. 20).

ABC50

Particularly preferred are human ABC50 homologous nucleic acids,particularly nucleic acids encoding a human ABC50 protein (TNF-alphastimulated ABC protein; GenBank Accession No. AF027302 for the cDNA,AAC70891 for the protein) and homologous genes of Drosophila ABC50(GadFly Accession Number CG1703), and rat ABC50 (GenBank Accession No.AF293383 for the cDNA). An alignment of ABC50 from different species hasbeen done by the ClustaW program.

Coronin

Particularly preferred are human coronin homologous nucleic acids,particularly nucleic acids encoding human actin-binding protein coronin1B (GenBank Accession No. NM_(—)020441 for the cDNA, NP_(—)065174 forthe protein, formerly GenBank Accession No. BC006449), humanactin-binding protein coronin 1C (GenBank Accession No. NM_(—)014325 forthe cDNA, NP_(—)055140 for the protein; GenBank Accession No. BC002342),human coronin protein (coronin homologue; GenBank Accession No. X89109for the cDNA, CAA61482 for the protein), human clipinE/coronin 6type B(see FIG. 27; Seq ID NO: 8), human Coronin 2A (GenBank Accession No.Q92828 for the protein), and human Coronin 2B (GenBank Accession No.Q9UQ03 for the protein). An alignment of coronin from different specieshas been done by the ClustaW program (see also FIG. 29).

Sec61alpha

Particularly preferred are human Sec61 alpha homologous nucleic acids,particularly nucleic acids encoding a human Sec61 alpha form 2 protein(GenBank Accession No. NM_(—)018144 for the cDNA, NP_(—)060614 for theprotein, formerly GenBank Accession No. AF346603) and human Sec61 alphaform 1 protein (GenBank Accession No. NM_(—)013336.2 for the cDNA,NP_(—)037468 for the protein, formerly AF346602). An alignment of Sec61alpha from different species has been done by the ClustaW program.Drosophila Sec61 alpha is also homologous to mouse Sec61 alpha-2 protein(GenBank Accession No. AF222748) and mouse Sec61 isoform 1 protein(GenBank Accession No. AF145253).

vATPase

Particularly preferred are nucleic acids encoding a Drosophila VhaPPA1-1(GadFly Accession Number CG7007), human ATPase, H+transporting,lysosomal 21 kD (vacuolar protein pump) protein (GenBank Accession No.NM_(—)004047 for the cDNA, NP_(—)004038 for the protein), and mouseATPase, H+transporting, lysosomal 21 kD (vacuolar protein pump) protein(GenBank Accession No. NM_(—)033617 for the cDNA). An alignment ofVhaPPA1-1 from different species has been done by the ClustaW program. Acomparison between the Drosophila and the human vacuolar ATPase (GenBankAccession No. NP_(—)004038) shows 63% identity (124 of 194 amino acids)and 76% similarity (150 of 194 amino acids).

EXAMPLE 4 Expression of the Polypeptides in Mammalian (Mouse) Tissues

For analyzing the expression of the polypeptides disclosed in thisinvention in mammalian tissues, several mouse strains (preferrably micestrains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db, which are standardmodel systems in obesity and diabetes research) were purchased fromHarlan Winkelmann (33178 Borchen, Germany) and maintained under constanttemperature (preferrably 22° C.), 40% humidity and a light/dark cycle ofpreferrably 14/10 hours. The mice were fed a standard chow (for example,from ssniff Spezialitaten GmbH, order number ssniff M-Z V1126-000). Forthe fasting experiment (“fasted wild type mice”), wild type mice werestarved for 48 h without food, but only water supplied ad libitum. (see,for example, Schnetzler et al. J Clin Invest 1993 July;92(1):272-80,Mizuno et al. Proc Natl Acad Sci U S A 1996 April 16;93(8):3434-8).Animals were sacrificed at an age of 6 to 8 weeks. Male mice(preferrably mouse strain C57BL/6) were placed at the age of 4 weeks ingroups of 8 animals (N=8) for 16 weeks on control diet (preferablyAltromin C1057 mod control, 4.5% crude fat, or high fat diet (preferablyAltromin C1057 mod. high fat, 23.5% crude fat). At the age of 20 weeksmice were sacrified and different tissues and organs dissected. Theanimal tissues and organs were isolated according to standard proceduresknown to those skilled in the art, snap frozen in liquid nitrogen andstored at −80° C. until needed.

For analyzing the role of the proteins disclosed in this invention inthe in vitro differentiation of different mammalian cell culture cellsfor the conversion of pre-adipocytes to adipocytes, mammalian fibroblast(3T3-L1) cells (e.g., Green & Kehinde, Cell 1: 113-116, 1974) wereobtained from the American Tissue Culture Collection (ATCC, Hanassas,Va., USA; ATCC-CL 173). 3T3-L1 cells were maintained as fibroblasts anddifferentiated into adipocytes as described in the prior art (e.g., Qiu.et al., J. Biol. Chem. 276:11988-95, 2001; Slieker et al., BBRC 251:225-9, 1998). At various time points of the differentiation procedure,beginning with day 0 (day of confluence) and day 2 (hormone addition;for example, dexamethason and 3-isobutyl-1-methylxanthin), up to 10 daysof differentiation, suitable aliquots of cells were taken every twodays. Alternatively, mammalian fibroblast 3T3-F442A cells (e.g., GreenH. and Kehinde O., (1976) Cell 7(1): 105-113) were obtained from theHarvard Medical School, Department of Cell Biology (Boston, Mass., USA).Alternatively, mammalian fibroblast TA1 cells (Chapman A. B. et al.,(1984) J Biol Chem 259(24):15548-15555) were obtained from ATCC.3T3-F442A and TA1 cells were maintained as fibroblasts anddifferentiated into adipocytes as described previously (Djian, P. etal., (1985) J. Cell. Physiol. 124 (3):554-556). At various time pointsof the differentiation procedure, beginning with day 0 (day ofconfluence and hormone addition, for example, Insulin), up to 10 days ofdifferentiation, suitable aliquots of cells were taken every two days.3T3-F442A cells and TA1cells are differentiating in vitro already in theconfluent stage after hormone (insulin) addition.

RNA was isolated from mouse tissues or cell culture cells using TrizolReagent (for example, from Invitrogen, Karlsruhe, Germany) and furtherpurified with the RNeasy Kit (for example, from Qiagen, Germany) incombination with an DNase-treatment according to the instructions of themanufacturers and as known to those skilled in the art. Total RNA wasreverse transcribed (preferrably using Superscript II RNaseH-ReverseTranscriptase, from Invitrogen, Karlsruhe, Germany) and subjected toTaqman analysis preferrably using the Taqman 2xPCR Master Mix (fromApplied Biosystems, Weiterstadt, Germany; the Mix contains according tothe Manufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG,dNTPs with dUTP, passive reference Rox and optimized buffer components)on a GeneAmp 5700 Sequence Detection System (from Applied Biosystems,Weiterstadt, Germany).

For the analysis of the expression of the proteins of the invention,taqman analysis was performed using the following primer/probe pairs:

-   Mouse Men forward primer (Seq ID NO: 9) 5′-GGG AGA CCT TGG CTG TAA    TGG-3′;-   Mouse Men reverse primer (Seq ID NO: 10) 5′-ACC CCT CCA CAT GCC    GT-3′;-   Mouse Men Taqman probe (Seq ID NO: 11) (5/6-FAM) CAT CCC TGT GGG TAA    ACT GGC CCT TT (5/6-TAMRA)-   Mouse m2GST2 forward primer (Seq ID NO: 12) 5′-CAA GCC AAC TCT TCC    ATT TGG-3′;-   Mouse m2GST2 reverse primer (Seq ID NO: 13) 5′-ATT GCG AGG CTC TGG    TGG-3′;-   Mouse m2GST2 Taqman probe (Seq ID NO: 14) (5/6-FAM) ATC CCT GTT TTG    GAG GTG GAA GGA CTT ACA (5/6-TAMRA)-   Mouse Rab32 forward primer (Seq ID NO: 15) 5′-GGT CCC AGT GCT GCT    GAT GT-3′;-   Mouse Rab32 reverse primer (Seq ID NO: 16) 5′-CCT CCA TAC AGG CAG    GAC CA-3′;-   Mouse Rab32 Taqman probe (Seq ID NO: 17) (5/6-FAM) TCT CTG TGC CCC    ATG TGC TGT CTC C (5/6-TAMRA)-   Mouse Rab38 forward primer (Seq ID NO: 18) 5′-ACC TCA CAA GGA GCA    CCT GTA CA-3′;-   Mouse Rab38 reverse primer (Seq ID NO: 19) 5′-TAA TGC TGG TCT TGC    CCA CA-3′;-   Mouse Rab38 Taqman probe (Seq ID NO: 20) (5/6-FAM) TGC TGG TGA TCG    GCG ACC TGG (5/6-TAMRA)-   Mouse Csp forward primer (Seq ID NO: 21) 5′-GGC ACA GCT GCA GTC TGA    TG-3′;-   Mouse Csp reverse primer (Seq ID NO: 22) 5′-TGG CAG ATG CTG GCT GTA    TG-3′;-   Mouse Csp Taqman probe (Seq ID NO: 23) (5/6-FAM) AAG GGA GGC TAC AGA    CAC ACC GAT CG (5/6-TAMRA)-   Mouse F-box forward primer (Seq ID NO: 24) 5′-CGT CGC CAG ACC CTG    ATT-3′;-   Mouse F-box reverse primer (Seq ID NO: 25) 5′-CAA ACG GCG GCT    CCC-3′;-   Mouse F-box Taqman probe (Seq ID NO: 26) (5/6-FAM) CAC AGT CCG AGA    CGT CAA ACT CCT GGT (5/6-TAMRA)-   Mouse ABC50 forward primer (Seq ID NO: 27) 5′-TCG ACA TGG ACT CCC    GGA T-3′;-   Mouse ABC50 reverse primer (Seq ID NO: 28) 5′-CAG GAG TAG TGT GCT    CTT CCC C-3′;-   Mouse ABC50 Taqman probe (Seq ID NO: 29) (5/6-FAM) TGC ATC GTG GGT    CCC AAT GGT G (516-TAMRA)-   Mouse Coronin 1B forward primer (Seq ID NO: 30) 5′-AGG GAC CAT CTC    CTC GAC CT-3′;-   Mouse Coronin 1B reverse primer (Seq ID NO: 31) 5′-CCC ATC TCT GCT    GCT TTT TCT G-3′;-   Mouse Coronin 1B Taqman probe (Seq ID NO: 32) (5/6-FAM) CCC AAC CCA    CTG CCC CCT CA (5/6-TAMRA)-   Mouse Coronin 1C forward primer (Seq ID NO: 33) 5′-CCG CGC ACT    CCCAGG-3′;-   Mouse Coronin 1C reverse primer (Seq ID NO: 34) 5′-CAA ATC TGA CAT    GGA ATG TCT CCA-3′;-   Mouse Coronin 1C Taqman probe (Seq ID NO: 35) (5/6-FAM) AGG GCA GAG    AGG GAG ACA CTG CCA (5/6-TAMRA)-   Mouse Coronin 6 forward primer (Seq ID NO: 36) 5′-TGA GAC CCA TGC    GGG CT-3′;-   Mouse Coronin 6 reverse primer (Seq ID NO: 37) 5′-TCG GGT GAA TCC    CGT GG-3′;-   Mouse Coronin 6 Taqman probe (Seq ID NO: 38) (516-FAM) TCT TCA CGC    GGC TGG GTC ATA TCT TC (5/6-TAMRA)-   Mouse vATPaseVO forward primer (Seq ID NO: 39) 5′-GGC TTG GTG TTC    AGG GTC TC-3′;-   Mouse vATPaseVO reverse primer (Seq ID NO: 40) 5′-ACT GCA ATG CCT    CCA GAG TCA-3′;-   Mouse vATPaseVO Taqman probe (Seq ID NO: 41) (5/6-FAM) CCT GCA CTC    ACC TCT TGC TGC CTG (5/6-TAMRA)

The results of the real time PCR (Taqman) analysis are shown in theFIGS. 4, 7, 11, 16, 21, 24, 30, and 36.

Men

As shown in FIG. 4A, real time PCR (Taqman) analysis of the expressionof the Men protein in mammalian (mouse) tissues revealed that Men isexpressed in different mammalian tissues, showing higher levels ofexpression in BAT, testis, kidney, liver, and WAT tissues. With regardto changes in expression intensity during the differentiation ofpreadipocytes to adipocytes, a strong increase in relative signalintensity can be observed for Men during the in vitro differentiationprogram of 3T3-L1 cells (see

FIG. 4B). The results of both experiments show that Men plays a role inthe metabolism of adipose tissue and therefore suggests a relevance ofthis gene for metabolic disorders.

Gst2

The expression of the m2GST2 is strongly upregulated in WAT of bothanimal models of obesity used in these experiments. In two more tissueswhich are highly relevant for metabolic disorders, namely BAT and musclethe expression of m2GST2 is also upregulated in both models. Theseexpression patterns strongly suggest that m2GST2 has an essentialfunction in adipose tissue and muscle. In contrast m2GST-2 was notexpressed in the 3T3-L1 adipocte cell line (Data not shown). As m2GST-2is most likely involved in the synthesis of signalling molecules it islikely that its expression is under the control of external stimuli,which are not present in the cell culture system used. An indication forthis is the strong response observed in our animal models.

Rab32 and Rab38

Taqman analysis revealed that Rab32 and Rab38 are equally interestinghomologues of the fly gene. Both are rather ubiquitously expressed withRab38 showing a stronger expression in lung, spleen and kidney (FIG.11B). Both genes show an upregulation of their expression in WAT and BATof genetically ob/ob mice (FIGS. 11C and 11D). A further example of theregulation of these genes under different metabolic conditions isprovided by their marked downregulation in WAT, BAT and muscle of fastedmice. In addition, a significant upregulation in kidney of fasted miceis noted (FIGS. 11C and 11D). The upregulation of Rab32 and Rab38 isalso observed in WAT, BAT and heart of the genetically obese db/db mice(FIGS. 11E and 11F). Expression of Rab32 is induced during the in vitrodifferentiation of 3T3-L1 cells from preadipocytes to adipocytes (FIG.11G).

Csp

Taqman analysis revealed that CSP is consistently upregulated during thein vitro differentiation of preadipocytes to adipocytes (FIGS. 16A, 16Band 16C).

F-Box

Taqman analysis revealed that F-Box is rather ubiquitously expressed(FIG. 21A). F-Box expression is under metabolic control: In fasted as towell as obese (db/db) mice, expression is increased in brown adiposetissue (FIGS. 21B and 21C). In addition, expression of F-Box is stronglyinduced in BAT, liver and small intestine in mice hold under a high fatdiet (FIG. 21D). During the in vitro differentiation of 3T3-L1 as wellas of two additional model systems for the in vitro differentiation ofpreadipocytes to adipocytes, the 3T3-F442A and TA1, cell lines, theexpression of F-Box is dramatically reduced (FIGS. 21E, 21F, and 21G).

ABC50

As shown in FIG. 24A, real time PCR (Taqman) analysis of the expressionof the ABC50 protein in mouse tissues revealed that ABC50 is expressedin different mammalian tissues, showing higher levels of expression intestis, spleen, heart, hypothalamus, and muscle tissues. With regard tochanges in expression intensity during the differentiation ofpreadipocytes to adipocytes, Taqman analysis revealed a consistentlyupregulated expression of ABC50 during the in vitro differentiation ofpreadipocytes to adipocytes (see FIGS. 24B, 24C, and 24D).

Coronin

Taqman analysis revealed that Coronin 1C is the more interestinghomologue of the fly gene. In comparison to Coronin 6, which isrestricted to muscle and heart, Coronin 1B and 1C are ubiquitouslyexpressed with clear expression in WAT and BAT (FIGS. 30A, 30B, and30C). The expression of Coronin 1C in white and brown adipose tissue isunder metabolic control: in genetically obese (ob/ob) mice, expressionof Coronin 1C is strongely induced in these tissues compared to wildtypelevels (FIG. 30D).

vATPase

Taqman analysis revealed that vATPaseVO shows a clear upregulation ofits expression intensity during the differentiation of preadipocytes toadipocytes (FIGS. 36A, 36B, and 36C).

All publications and patents mentioned in the above specification areherein incorporated by reference.

Various modifications and variations of the described method and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1. A pharmaceutical composition comprising a nucleic acid molecule ofthe Men (malic enzyme) gene family, the GST2 (glutathione S-transferase2) gene family, the Rab-RP1 family of proteins, the cystein stringprotein (Csp) family of proteins, the F-box gene family, the ABC50 genefamily, the coronin family of actin-associated proteins, the Sec61 alphagene family, or the vacuolar ATPase gene family or a polypeptide encodedthereby or a fragment or a variant of said nucleic acid molecule or saidpolypeptide or an antibody, an aptamer or another receptor recognizing anucleic acid molecule of the Men, GST2, Rab-RP1, Csp, F-box proteinLilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1 gene family or apolypeptide encoded thereby together with pharmaceutically acceptablecarriers, diluents and/or adjuvants.
 2. The composition of claim 1,wherein the nucleic acid molecule is a vertebrate or insect Men protein,GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61alpha, or VhaPPA1-1 nucleic acid, particularly encoding human Menproteins (malic enzyme 1; GenBank Accession No. NM_(—)002395 for thecDNA, NP_(—)002386 for the protein, malic enzyme 3; NM_(—)006680 for thecDNA, NP_(—)006671 for the protein, malic enzyme 2; GenBank AccessionNo. NM_(—)002396.2 for the cDNA, NP_(—)002387 for the protein), a humanGST2 protein (hematopoietic prostaglandin D2 synthase; GenBank AccessionNo. NM_(—)014485 for the cDNA, NP_(—)055300 for the protein), a mouseGST2 protein (hematopoietic prostaglandin D2 synthase 2; GenBankAccession No. NM_(—)019455 for the cDNA, NP_(—)062328 for the protein),a rat GST2 protein (hematopoietic prostaglandin D2 synthase 2; GenBankAccession No. NM_(—)031644 for the cDNA, NP_(—)113832 for the protein),human Rab-RP1 proteins (Rab32; GenBank Accession No. NM_(—)006834 forthe cDNA, NP_(—)006825 for the protein, formerly GenBank Accession No.XM_(—)004076, Rab38; GenBank Accession No. NM_(—)022337 for the cDNA,NP_(—)071732 for the protein, formerly XM_(—)015771, Rab7; GenBankAccession No. NM_(—)003929 for the cDNA, NP_(—)003920 for the protein),mouse Rab-RP1 proteins (Rab32; GenBank Accession No. NM_(—)026405 forthe cDNA, NP_(—)080681 for the protein, Rab38; GenBank Accession No.NM_(—)028238 for the cDNA, NP_(—)082514 for the protein), human Cspproteins (Csp; EnsEMBL Accession No. ENST00000217123 (SEQ ID NO. 7) forthe cDNA, GenBank Accession No. CAC15495.1 for the protein, Csp1;GenBank Accession No. S70515 for the protein, gamma Csp; GenBankAccession No. AK097736 for the cDNA, BAC05155 for the protein, Beta Csp;GenBank Accession No. Q9UF47 for the protein), human F-box Lilina/FBL7proteins (F-box and leucine-rich repeat protein 11; GenBank AccessionNo. NM_(—)012308 for the cDNA, NP_(—)036440 for the protein, JEMMAprotein; GenBank Accession No. CAD30700 for the protein, PDH fingerprotein 2; GenBank Accession Number NM_(—)005392 for the cDNA,NP_(—)005383 for the protein, protein similar to several hypotheticalproteins; GenBank Accession No. AAC83407 for the protein), a human ABC50protein (TNF-alpha stimulated ABC protein; GenBank Accession No.AF027302 for the cDNA, AAC70891 for the protein), a rat ABC50 protein(GenBank Accession No. AF293383 for the cDNA, AAG23960 for the protein),human coronin proteins (actin-binding protein coronin 1B; GenBankAccession No. NM_(—)020441 for the cDNA, NP_(—)065174 for the protein,formerly BC006449, actin-binding protein coronin 1C; GenBank AccessionNo. NM_(—)014325 for the cDNA, NP_(—)055140 for the protein; formerlyBC002342, coronin homologue; GenBank Accession No. X89109 for the cDNA,CAA61482 for the protein, clipinE/coronin 6 type B; Seq ID NO: 8,Coronin 2A; GenBank Accession No. Q92828 for the protein, Coronin 2B;GenBank Accession No. Q9UQ03 for the protein), human Sec61 alphaproteins (Sec61 alpha form 2 protein; GenBank Accession No. NM_(—)018144for the cDNA, NP_(—)060614 for the protein, formerly AF346603, humanSec61 alpha form 1 protein; GenBank Accession No. NM_(—)013336.2 for thecDNA, NP_(—)037468 for the protein, formerly AF346602), mouse Sec61alpha proteins (Sec61 alpha-2 protein; GenBank Accession No. AF222748for the cDNA, AAG44253 for the protein, Sec61 alpha isoform 1 protein;GenBank Accession No. AF145253 for the cDNA. AAF66695 for the protein)or a human VhaPPA1-1 protein (ATPase, H+transporting, lysosomal 21 kD(vacuolar protein pump) protein; GenBank Accession No. NM_(—)004047 forthe cDNA, NP_(—)004038 for the protein), or mouse VhaPPA1-1 protein(ATPase, H+transporting, lysosomal 21 kD protein; GenBank Accession No.NM_(—)033617 for the cDNA, NP_(—)291095 for the protein), or aDrosophila Men protein (GadFly Accession Number CG10120), a DrosophilaGST2 protein (GstS1; GadFly Accession Number CG8938), a DrosophilaRab-RP1 protein (GadFly Accession Number CG8024), a Drosophila Cspprotein (GadFly Accession Number CG6395), a Drosophila F-box proteinLilina/FBL7 (GadFly Accession Number CG11033), a Drosophila ABC50protein (GadFly Accession Number CG1703), a Drosophila coro protein(GadFly Accession Number CG9446), a Drosophila sec61 alpha protein(GadFly Accession Number CG9539), or a Drosophila VhaPPA1-1 protein(GadFly Accession Number CG7007), or a fragment thereof or a variantthereof and/or a nucleic acid molecule complementary thereto.
 3. Thecomposition of claim 1, wherein said nucleic acid molecule (a)hybridizes at 50□C in a solution containing 1×SSC and 0.1% SDS to anucleic acid molecule as defined in claim 2 and/or a nucleic acidmolecule which is complementary thereto; (b) it is degenerate withrespect to the nucleic acid molecule of (a) (c) encodes a polypeptidewhich is at least 85%, preferably at least 90%, more preferably at least95%, more preferably at least 98% and up to 99.6% identical to thepolypeptides as defined in claim 2; (d) differs from the nucleic acidmolecule of (a) to (c) by mutation and wherein said mutation causes analteration, deletion, duplication or premature stop in the encodedpolypeptide.
 4. The composition of claim 1, wherein the nucleic acidmolecule is a DNA molecule, particularly a cDNA or a genomic DNA.
 5. Thecomposition of claim 1, wherein said nucleic acid encodes a polypeptidecontributing to regulating the energy homeostasis and/or the metabolismof triglycerides.
 6. The composition of claim 1, wherein said nucleicacid molecule is a recombinant nucleic acid molecule.
 7. The compositionof any claim 1, wherein the nucleic acid molecule is a vector,particularly an expression vector.
 8. The composition of claim 1,wherein the polypeptide is a recombinant polypeptide.
 9. The compositionof claim 8, wherein said recombinant polypeptide is a fusionpolypeptide.
 10. The composition of claim 1, wherein said nucleic acidmolecule is selected from hybridization probes, primers and anti-senseoligonucleotides.
 11. The composition of claim 1 which is a diagnosticcomposition.
 12. The composition of claim 1 which is a therapeuticcomposition.
 13. The composition of claim 1 for the manufacture of anagent for detecting and/or verifying, for the treatment, alleviationand/or prevention of an disorders, including metabolic diseases such asobesity and other body-weight regulation disorders as well as relateddisorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, and sleep apnea and others, in cells, cell masses,organs and/or subjects.
 14. Use of a nucleic acid molecule of the Men,GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61alpha, or VhaPPA1-1 gene family or a polypeptide encoded thereby or afragment or a variant of said nucleic acid molecule or said polypeptideor an antibody, an aptamer or another receptor recognizing a nucleicacid molecule of the Men, GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7,ABC50, coronin, Sec61 alpha, or VhaPPA1-1 gene family or a polypeptideencoded thereby for controlling the function of a gene and/or a geneproduct which is influenced and/or modified by a Men, GST2, Rab-RP1,Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, orVhaPPA1-1 homologous polypeptide.
 15. Use of the nucleic acid moleculeof the Men, GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50,coronin, Sec61 alpha, or VhaPPA1-1 gene family or a polypeptide encodedthereby or a fragment or a variant of said nucleic acid molecule or saidpolypeptide or an antibody, an aptamer or another receptor recognizing anucleic acid molecule of the Men, GST2, Rab-RP1, Csp, F-box proteinLilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1 protein genefamily or a polypeptide encoded thereby for identifying substancescapable of interacting with a Men, GST2, Rab-RP1, Csp, F-box proteinLilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1 homologouspolypeptide.
 16. A non-human transgenic animal exhibiting a modifiedexpression of a Men, GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7,ABC50, coronin, Sec61 alpha, or VhaPPA1-1 homologous polypeptide. 17.The animal of claim 16, wherein the expression of the Men, GST2,Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, orVhaPPA1-1 homologous polypeptide is increased and/or reduced.
 18. Arecombinant host cell exhibiting a modified expression of a Men, GST2,Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, orVhaPPA1-1 homologous polypeptide.
 19. The cell of claim 18 which is ahuman cell.
 20. A method of identifying a (poly)peptide involved in theregulation of energy homeostasis and/or metabolism of triglycerides in amammal comprising the steps of (a) contacting a collection of(poly)peptides with a Men, GST2, Rab-RP1, Csp, F-box proteinLilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1 homologouspolypeptide or a fragment thereof under conditions that allow binding ofsaid (poly)peptides; (b) removing (poly)peptides which do not bind and(c) identifying (poly)peptides that bind to said Men, GST2, Rab-RP1,Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, orVhaPPA1-1 homologous polypeptide.
 21. A method of screening for an agentwhich modulates the interaction of a Men, GST2, Rab-RP1, Csp, F-boxprotein Lilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1homologous polypeptide with a binding target/agent, comprising the stepsof (a) incubating a mixture comprising (aa) a Men, GST2, Rab-RP1, Csp,F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1homologous polypeptide, or a fragment thereof; (ab) a bindingtarget/agent of said Men, GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7,ABC50, coronin, Sec61 alpha, or VhaPPA1-1 homologous polypeptide orfragment thereof; and (ac) a candidate agent under conditions wherebysaid Men, GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin,Sec61 alpha, or VhaPPA1-1 polypeptide or fragment thereof specificallybinds to said binding target/agent at a reference affinity; (b)detecting the binding affinity of said Men, GST2, Rab-RP1, Csp, F-boxprotein Lilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1polypeptide or fragment thereof to said binding target to determine a(candidate) agent-biased affinity; and (c) determining a differencebetween (candidate) agent-biased affinity and reference affinity.
 22. Amethod of screening for an agent which modulates the activity of a Men,GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61alpha, or VhaPPA1-1 homologous polypeptide comprising the steps of (a)incubating a mixture comprising (aa) a Men, GST2, Rab-RP1, Csp, F-boxprotein Lilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1homologous polypeptide, or a fragment thereof and (ab) a candidate agentunder conditions whereby said Men, GST2, Rab-RP1, Csp, F-box proteinLilina/FBL7, ABC50, coronin, Sec61 alpha, or VhaPPA1-1 polypeptide orfragment thereof exhibits a reference activity; (b) detecting theactivity of said Men, GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7,ABC50, coronin, Sec61 alpha, or VhaPPA1-1 polypeptide or fragmentthereof to determine a (candidate) agent-biased activity; and (c)determining a difference between (candidate) agent-biased activity andreference activity.
 23. (Cancelled)
 24. (Cancelled)
 25. Use of a(poly)peptide as identified by the method of claim 20 for thepreparation of a pharmaceutical composition for the treatment,alleviation and/or prevention of diseases and disorders, includingmetabolic diseases such as obesity and other body-weight regulationdisorders as well as related disorders such as eating disorder,cachexia, diabetes mellitus, hypertension, coronary heart disease,hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, cancer,e.g. cancers of the reproductive organs, and sleep apnea and otherdiseases and disorders.
 26. Use of a nucleic acid molecule of the Men,GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61alpha, or VhaPPA1-1 family or of a fragment thereof for the preparationof a non-human animal which over- or under-expresses the Men, GST2,Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61 alpha, orVhaPPA1-1 gene product.
 27. Kit comprising at least one of (a) a Men,GST2, Rab-RP1, Csp, F-box protein Lilina/FBL7, ABC50, coronin, Sec61alpha, or VhaPPA1-1 nucleic acid molecule or a fragment thereof; (b) avector comprising the nucleic acid of (a); (c) a host cell comprisingthe nucleic acid of (a) or the vector of (b); (d) a polypeptide encodedby the nucleic acid of (a); (e) a fusion polypeptide encoded by thenucleic acid of (a); (f) an antibody, an aptamer or another receptoragainst the nucleic acid of (a) or the polypeptide of (d) or (e) and (g)an anti-sense oligonucleotide of the nucleic acid of (a).
 28. Use of anagent as identified by the method of claim 21 for the preparation of apharmaceutical composition for the treatment, alleviation and/orprevention of diseases and disorders, including metabolic diseases suchas obesity and other body-weight regulation disorders as well as relateddisorders such as eating disorder, cachexia, diabetes mellitus,hypertension, coronary heart disease, hypercholesterolemia,dyslipidemia, osteoarthritis, gallstones, cancer, e.g. cancers of thereproductive organs, and sleep apnea and other diseases and disorders.